Modified nucleotides

ABSTRACT

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure —O—Z wherein Z is any of —C(R′)2-O—R″, —C(R′)2-N(R″)2, —C(R′)2-N(H)R″, —C(R′)2-S—R″ and —C(R′)2-F, wherein each R″ is or is part of a removable protecting group; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R′)2 represents an alkylidene group of formula ═C(R′″)2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —C(R′)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′OH; with the proviso that where Z is —C(R′)2-S—R″, both R′ groups are not H.

The invention relates to modified nucleotides. In particular, thisinvention discloses nucleotides having a removable protecting group,their use in polynucleotide sequencing methods and a method for chemicaldeprotection of the protecting group.

Advances in the study of molecules have been led, in part, byimprovement in technologies used to characterise the molecules or theirbiological reactions. In particular, the study of the nucleic acids DNAand RNA has benefited from developing technologies used for sequenceanalysis and the study of hybridisation events.

An example of the technologies that have improved the study of nucleicacids is the development of fabricated arrays of immobilised nucleicacids. These arrays consist typically of a high-density matrix ofpolynucleotides immobilised onto a solid support material. See, e.g.,Fodor et al., Trends Biotech. 12:19-26, 1994, which describes ways ofassembling the nucleic acids using a chemically sensitized glass surfaceprotected by a mask, but exposed at defined areas to allow attachment ofsuitably modified nucleotide phosphoramidites. Fabricated arrays canalso be manufactured by the technique of “spotting” knownpolynucleotides onto a solid support at predetermined positions (e.g.,Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383, 1995).

Sequencing by synthesis of DNA ideally requires the controlled (i.e. oneat a time) incorporation of the correct complementary nucleotideopposite the oligonucleotide being sequenced. This allows for accuratesequencing by adding nucleotides in multiple cycles as each nucleotideresidue is sequenced one at a time, thus preventing an uncontrolledseries of incorporations occurring. The incorporated nucleotide is readusing an appropriate label attached thereto before removal of the labelmoiety and the subsequent next round of sequencing. In order to ensureonly a single incorporation occurs, a structural modification (“blockinggroup”) of the sequencing nucleotides is required to ensure a singlenucleotide incorporation but which then prevents any further nucleotideincorporation into the polynucleotide chain. The blocking group mustthen be removable, under reaction conditions which do not interfere withthe integrity of the DNA being sequenced. The sequencing cycle can thencontinue with the incorporation of the next blocked, labellednucleotide. In order to be of practical use, the entire process shouldconsist of high yielding, highly specific chemical and enzymatic stepsto facilitate multiple cycles of sequencing.

To be useful in DNA sequencing, nucleotide, and more usually nucleotidetriphosphates, generally require a 3′OH-blocking group so as to preventthe polymerase used to incorporate it into a polynucleotide chain fromcontinuing to replicate once the base on the nucleotide is added. Thereare many limitations on the suitability of a molecule as a blockinggroup. It must be such that it prevents additional nucleotide moleculesfrom being added to the polynucleotide chain whilst simultaneously beingeasily removable from the sugar moiety without causing damage to thepolynucleotide chain. Furthermore, the modified nucleotide must betolerated by the polymerase or other appropriate enzyme used toincorporate it into the polynucleotide chain. The ideal blocking groupwill therefore exhibit long term stability, be efficiently incorporatedby the polymerase enzyme, cause total blocking of secondary or furtherincorporation and have the ability to be removed under mild conditionsthat do not cause damage to the polynucleotide structure, preferablyunder aqueous conditions. These stringent requirements are formidableobstacles to the design and synthesis of the requisite modifiednucleotides.

Reversible blocking groups for this purpose have been describedpreviously but none of them generally meet the above criteria forpolynucleotide, e.g. DNA-compatible, chemistry.

Metzker et al., (Nucleic Acids Research, 22(20): 4259-4267, 1994)discloses the synthesis and use of eight 3′-modified2-deoxyribonucleoside 5′-triphosphates (3′-modified dNTPs) and testingin two DNA template assays for incorporation activity. The 3′-modifieddNTPs included 3′allyl deoxyriboadenosine 5′-triphosphate (3′-allyldATP). However, the 3′allyl blocked compound was not used to demonstratea complete cycle of termination, deprotection and reinitiation of DNAsynthesis: the only test results presented were those which showed theability of this compound to terminate DNA synthesis in a singletermination assay, out of eight such assays conducted, each conductedwith a different DNA polymerase.

WO02/29003 (The Trustees of Columbia University in the City of New York)describes a sequencing method which, may include the use of an allylprotecting group to cap the 3′-OH group on a growing strand of DNA in apolymerase reaction. The allyl group is introduced according to theprocedure of Metzker (infra) and is said to be removed by usingmethodology reported by Kamal et al (Tet. Let, 40, 371-372, 1999).

The Kamal deprotection methodology employs sodium iodide andchlorotrimethylsilane so as to generate in situ iodotrimethylsilane, inacetonitrile solvent, quenching with sodium thiosulfate. Afterextraction into ethyl acetate and drying (sodium sulfate), thenconcentration under reduced pressure and column chromatography (ethylacetate:hexane; 2:3 as eluant), free alcohols were obtained in 90-98%yield.

In WO02/29003, the Kamal allyl deprotection is suggested as beingdirectly applicable in DNA sequencing without modification, the Kamalconditions being mild and specific.

While Metzker reports on the preparation of a 3′allyl-blocked nucleotideor nucleoside and WO02/29003 suggests the use of the allyl functionalityas a 3′-OH cap during sequencing, neither of these documents actuallyteaches the deprotection of 3′-allylated hydroxyl group in the contextof a sequencing protocol. Whilst the use of an allyl group as a hydroxylprotecting group is well known—it is easy to introduce and is stableacross the whole pH range and to elevated temperatures—there is to date,no concrete embodiment of the successful cleavage of a 3′-allyl groupunder DNA compatible conditions, i.e. conditions under which theintegrity of the DNA is not wholly or partially destroyed. In otherwords, it has not been possible hitherto to conduct DNA sequencing using3′OH allyl-blocked nucleotides.

The Kamal methodology is inappropriate to conduct in aqueous media sincethe TMS chloride will hydrolyse preventing the in situ generation of TMSiodide. Attempts to carry out the Kamal deprotection (in acetonitrile)in sequencing have proven unsuccessful in our hands.

The present invention is based on the surprising development of a numberof reversible blocking groups and methods of deprotecting them under DNAcompatible conditions. Some of these blocking groups are novel per se;others have been disclosed in the prior art but, as noted above, it hasnot proved possible to utilised these blocking groups in DNA sequencing.

One feature of the invention derives from the development of acompletely new method of allyl deprotection. Our procedure is of broadapplicability to the deprotection of virtually all allyl-protectedhydroxyl functionality and may be effected in aqueous solution, incontrast to the methodology of Kamal et al. (which is effected inacetonitrile) and to the other methods known generally in the prior artwhich are highly oxygen- and moisture-sensitive. A further feature ofthe invention derives from the development of a new class of protectinggroups. These are based upon acetals and related protecting groups butdo not suffer from some of the disadvantages of acetal deprotectionknown in the prior art.

The allyl deprotection methodology makes use of a water-solubletransition metal catalyst formed from a transition metal and at leastpartially water-soluble ligands. In aqueous solution these form at leastpartially water-soluble transition metal complexes. By aqueous solutionherein is meant a liquid comprising at least 20 vol %, preferably atleast 50%, for example at least 75 vol %, particularly at least 95 vol %and especially greater than above 98 vol %, ideally 100 vol % of wateras the continuous phase.

As those skilled in the art will appreciate, the allyl group may be usedto protect not only the hydroxyl group but also thiol and aminefunctionalities. Moreover allylic esters may be formed from the reactionbetween carboxylic acids and allyl halides, for example. Primary orsecondary amides may also be protected using methods known in the art.The novel deprotection methodology described herein may be used in thedeprotection of all these allylated compounds, e.g. allyl esters andmono- or bisallylated primary amines or allylated amides, or in thedeprotection of allylated secondary amines. The method is also suitablein the deprotection of allyl esters and thioethers.

Protecting groups which comprise the acetal functionality have been usedpreviously as blocking groups. However, removal of such groups andethers requires strongly acidic deprotections detrimental to DNAmolecules. The hydrolysis of an acetal however, results in the formationof an unstable hemiacetal intermediate which hydrolyses under aqueousconditions to the natural hydroxyl group. The inventors have utilisedthis concept and applied it further such that this feature of theinvention resides in utilising blocking groups that include protectinggroups to protect intermediate molecules that would normally hydrolyseunder aqueous conditions. These protecting groups comprise a secondfunctional group that stabilises the structure of the intermediate butwhich can be removed at a later stage following incorporation into thepolynucleotide. Protecting groups have been used in organic synthesisreactions to temporarily mask the characteristic chemistry of afunctional group because it interferes with another reaction.

Therefore, according to a first aspect of the invention there isprovided a modified nucleotide or nucleoside molecule comprising apurine or pyrimidine base and a ribose or deoxyribose sugar moietyhaving a removable 3′-OH blocking group covalently attached thereto,such that the 3′ carbon atom has attached a group of the structure

—O—Z

wherein Z is any of —C(R′)₂—O—R″, —C(R′)₂—N(R″)₂, —C(R′)₂—N(H)R″,—C(R′)₂—S—R″ and —C(R′)₂—F,

wherein each R″ is or is part of a removable protecting group;

each R′ is independently a hydrogen atom, an alkyl, substituted alkyl,arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl,cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectablelabel attached through a linking group; or (R′)₂ represents analkylidene group of formula ═C(R′″)₂ wherein each R′″ may be the same ordifferent and is selected from the group comprising hydrogen and halogenatoms and alkyl groups; and

wherein said molecule may be reacted to yield an intermediate in whicheach R″ is exchanged for H or, where Z is —C(R′)₂—F, the F is exchangedfor OH, SH or NH₂, preferably OH, which intermediate dissociates underaqueous conditions to afford a molecule with a free 3′OH;

with the proviso that where Z is —C(R′)₂—S—R″, both R′ groups are not H.

Viewed from another aspect, the invention provides a, 3′-O-allylnucleotide or nucleoside which nucleotide or nucleoside comprises adetectable label linked to the base of the nucleoside or nucleotide,preferably by a cleavable linker.

In a further aspect, the invention provides a polynucleotide comprisinga 3′-O-allyl nucleotide or nucleoside which nucleotide or nucleosidecomprises a detectable label linked to the base of the nucleoside ornucleotide, preferably by a cleavable linker.

Viewed from a still further aspect, the invention provides a method ofconverting a compound of formula R—O-allyl, R₂N(allyl), RNH(allyl),RN(allyl)₂ or R—S-allyl to a corresponding compound in which the allylgroup is removed and replaced by hydrogen, said method comprising thesteps of reacting a compound of formula R—O-allyl, R₂N(allyl),RNH(allyl), RN(allyl)₂ or R—S-allyl in aqueous solution with atransition metal comprising a transition metal and one or more ligandsselected from the group comprising water-soluble phosphine andwater-soluble nitrogen-containing phosphine ligands, wherein the or eachR is a water-soluble biological molecule.

In a further aspect the invention provides a method of controlling theincorporation of a nucleotide molecule complementary to the nucleotidein a target single-stranded polynucleotide in a synthesis or sequencingreaction comprising incorporating into the growing complementarypolynucleotide a molecule according to the invention, the incorporationof said molecule preventing or blocking introduction of subsequentnucleoside or nucleotide molecules into said growing complementarypolynucleotide.

In a further aspect, the invention provides a method for determining thesequence of a target single-stranded, polynucleotide, comprisingmonitoring the sequential incorporation of complementary nucleotides,wherein at least one incorporation, and preferably all of theincorporations is of a nucleotide according to the invention ashereinbefore described which preferably comprises a detectable labellinked to the base of the nucleoside or nucleotide by a cleavable linkerand wherein the identity of the nucleotide incorporated is determined bydetecting the label, said blocking group and said label being removedprior to introduction of the next complementary nucleotide.

From a further aspect, the invention provides a method for determiningthe sequence of a target single-stranded polynucleotide, comprising:

(a) providing a plurality of different nucleotides according to thehereinbefore described invention which nucleotides are preferably linkedfrom the base to a detectable label by a cleavable linker and whereinthe detectable label linked to each type of nucleotide can bedistinguished upon detection from the detectable label used for othertypes of nucleotides;

(b) incorporating the nucleotide into the complement of the targetsingle-stranded polynucleotide;

(c) detecting the label of the nucleotide of (b), thereby determiningthe type of nucleotide incorporated;

(d) removing the label of the nucleotide of (b) and the blocking group;and

(e) optionally repeating steps (b)-(d) one or more times;

thereby determining the sequence of a target single-strandedpolynucleotide.

Additionally, in another aspect, the invention provides a kit,comprising:

(a) a plurality of different individual nucleotides of the invention;and

(b) packaging materials therefor.

The nucleosides or nucleotides according to or used in the methods ofthe present invention comprise a purine or pyrimidine base and a riboseor deoxyribose sugar moiety which has a blocking group covalentlyattached thereto, preferably at the 3′O position, which renders themolecules useful in techniques requiring blocking of the 3′-OH group toprevent incorporation of additional nucleotides, such as for example insequencing reactions, polynucleotide synthesis, nucleic acidamplification, nucleic acid hybridisation assays, single nucleotidepolymorphism studies, and other such techniques.

Where the term “blocking group” is used herein in the context of theinvention, this embraces both the allyl and “Z” blocking groupsdescribed herein. However, it will be appreciated that, in the methodsof the invention as described and claimed herein, where mixtures ofnucleotides are used, these very preferably each comprise the same typeof blocking, i.e. allyl-blocked or “Z”-blocked. Where “Z”-blockednucleotides are used, each “Z” group will generally be the same group,except in those cases where the detectable label forms part of the “Z”group, i.e. is not attached to the base.

Once the blocking group has been removed, it is possible to incorporateanother nucleotide to the free 3′-OH group.

The molecule can be linked via the base to a detectable label by adesirable linker, which label may be a fluorophore, for example. Thedetectable label may instead, if desirable, be incorporated into theblocking groups of formula “Z”. The linker can be acid labile,photolabile or contain a disulfide linkage. Other linkages, inparticular phosphine-cleavable azide-containing linkers, may be employedin the invention as described in greater detail.

Preferred labels and linkages included those disclosed in WO 03/048387.

In the methods where nucleotides are incorporated, e.g. where theincorporation of a nucleotide molecule complementary to the nucleotidein a target single stranded polynucleotide is controlled in a synthesisor sequencing reaction of the invention, the incorporation of themolecule may be accomplished via a terminal transferase, a polymerase ora reverse transcriptase.

Preferably, the molecule is incorporated by a polymerase andparticularly from Thermococcus sp., such as 9° N. Even more preferably,the polymerase is a mutant 9° N A485L and even more preferably is adouble mutant Y409V and A485L.

In the methods for determining the sequence of a target single-strandedpolynucleotide comprising monitoring the sequential incorporation ofcomplementary nucleotides of the invention, it is preferred that theblocking group and the label may be removed in a single chemicaltreatment step. Thus, in a preferred embodiment of the invention, theblocking group is cleaved simultaneously with the label. This will ofcourse be a feature inherent to those blocking groups of formula Z whichincorporate a detectable label.

Furthermore, preferably the blocked and labelled modified nucleotideconstructs of the nucleotide bases A, T, C and G are recognised assubstrates by the same polymerase enzyme.

In the methods described herein, each of the nucleotides can be broughtinto contact with the target sequentially, with removal ofnon-incorporated nucleotides prior to addition of the next nucleotide,where detection and removal of the label and the blocking group iscarried out either after addition of each nucleotide, or after additionof all four nucleotides.

In the methods, all of the nucleotides can be brought into contact withthe target simultaneously, i.e., a composition comprising all of thedifferent nucleotides is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label and the blocking group.

The methods can comprise a first step and a second step, where in thefirst step, a first composition comprising two of the four types ofmodified nucleotides is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label and the blocking group, and where inthe second step, a second composition comprising the two nucleotides notincluded in the first composition is brought into contact with thetarget, and non-incorporated nucleotides are removed prior to detectionand subsequent to removal of the label and blocking group, and where thefirst steps and the second step can be optionally repeated one or moretimes.

The methods described herein can also comprise a first step and a secondstep, where in the first step, a composition comprising one of the fournucleotides is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label and blocking group, and where in thesecond step, a second composition, comprising the three nucleotides notincluded in the first composition is brought into contact with thetarget, and non-incorporated nucleotides are removed prior to detectionand subsequent to removal of the label and blocking group, and where thefirst steps and the second step can be optionally repeated one or moretimes.

The methods described herein can also comprise a first step and a secondstep, where in the first step, a first composition comprising three ofthe four nucleotides is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label and blocking group and where in thesecond step, a composition comprising the nucleotide not included in thefirst composition is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label and blocking group, and where thefirst steps and the second step can be optionally repeated one or moretimes.

The incorporating step in the methods of the invention can beaccomplished via a terminal transferase, a polymerase or a reversetranscriptase as hereinbefore defined. The detectable label and/or thecleavable linker can be of a size sufficient to prevent theincorporation of a second nucleotide or nucleoside into the nucleic acidmolecule.

In certain methods described herein for determining the sequence of atarget single-stranded polynucleotide, each of the four nucleotides, oneof which will be complementary to the first unpaired base in the targetpolynucleotide, can be brought into contact with the targetsequentially, optionally with removal of non-incorporated nucleotidesprior to addition of the next nucleotide. Determination of the successof the incorporation may be carried out either after provision of eachnucleotide, or after the addition of all of the nucleotides added. If itis determined after addition of fewer than four nucleotides that one hasbeen incorporated, it is not necessary to provide further nucleotides inorder to detect the nucleotides complementary to the incorporatednucleotide.

Alternatively, all of the nucleotides can be brought into contact withthe target simultaneously, i.e., a composition comprising all of thedifferent nucleotide (i.e. A, T, C and G or A, U, C and G) is broughtinto contact with the target, and non-incorporated nucleotides removedprior to detection and removal of the label(s). The methods involvingsequential addition of nucleotides may comprise a first substep andoptionally one or more subsequent substeps. In the first substep acomposition comprising one, two or three of the four possiblenucleotides is provided, i.e. brought into contact with, the target.Thereafter any unincorporated nucleotides may be removed and a detectingstep may be conducted to determine whether one of the nucleotides hasbeen incorporated. If one has been incorporated, the cleavage of thelinker may be effected. In this way the identity of a nucleotide in thetarget polynucleotide may be determined. The nascent polynucleotide maythen be extended to determine the identity of the next unpairednucleotide in the target oligonucleotide.

If the first substep above does not lead to incorporation of anucleotide, or if this is not known, since the presence of incorporatednucleotides is not sought immediately after the first substep, one ormore subsequent substeps may be conducted in which some or all, of thosenucleotides not provided in the first substep are provided either, asappropriate, simultaneously or subsequently. Thereafter anyunincorporated nucleotides may be removed and a detecting step conductedto determine whether one of the classes of nucleotide has beenincorporated. If one has been incorporated, cleavage of the linker maybe effected. In this way the identity of a nucleotide in the targetpolynucleotide may be determined. The nascent polynucleotide may then beextended to determine the identity of the next unpaired nucleotide inthe target oligonucleotide. If necessary, a third and optionally afourth substep may be effected in a similar manner to the secondsubstep. Obviously, once four substeps have been effected, all fourpossible nucleotides will have been provided and one will have beenincorporated.

It is desirable to determine whether a type or class of nucleotide hasbeen incorporated after any particular combination comprising one, twoor three nucleotides has been provided. In this way the unnecessary costand time expended in providing the other nucleotide(s) is obviated. Thisis not a required feature of the invention, however.

It is also desirable, where the method for sequencing comprises one ormore substeps, to remove any unincorporated nucleotides before furthernucleotide are provided. Again, this is not a required feature of theinvention. Obviously, it is necessary that at least some and preferablyas many as practicable of the unincorporated nucleotides are removedprior to the detection of the incorporated nucleotide.

The kits of the invention include: (a) individual nucleotides accordingto the hereinbefore described invention, where each nucleotide has abase that is linked to a detectable label via a cleavable linker, or adetectable label linked via an optionally cleavable liner to a blockinggroup of formula Z, and where the detectable label linked to eachnucleotide can be distinguished upon detection from the detectable labelused for other three nucleotides; and (b) packaging materials therefor.The kit can further include an enzyme for incorporating the nucleotideinto the complementary nucleotide chain and buffers appropriate for theaction of the enzyme in addition to appropriate chemicals for removal ofthe blocking group and the detectable label, which can preferably beremoved by the same chemical treatment step.

The nucleotides/nucleosides are suitable for use in many differentDNA-based methodologies, including DNA synthesis and DNA sequencingprotocols.

The invention may be understood with reference to the attached drawingsin which:

FIG. 1 shows exemplary nucleotide structures useful in the invention.For each structure, X can be H, phosphate, diphosphate or triphosphate.R₁ and R₂ can be the same or different, and can be selected from H, OH,or any group which can be transformed into an OH, including, but notlimited to, a carbonyl. Some suitable functional groups for R₁ and R₂include the structures shown in FIG. 3 and FIG. 4.

FIG. 2 shows structures of linkers useful in certain aspects of theinvention, including (1) disulfide linkers and acid labile linkers, (2)dialkoxybenzyl linkers, (3) Sieber linkers, (4) indole linkers and (5)t-butyl Sieber linkers.

FIG. 3 shows some functional molecules useful in the invention,including some cleavable linkers and some suitable hydroxyl protectinggroups. In these structures, R₁ and R₂ may be the same of different, andcan be H, OH, or any group which can be transformed into an OH group,including a carbonyl. R₃ represents one or more substituentsindependently selected from alkyl, alkoxyl, amino or halogen groups. R₄and R₅ can be H or alkyl, and R₆ can be alkyl, cycloalkyl, alkenyl,cycloalkenyl or benzyl. X can be H, phosphate, diphosphate ortriphosphate.

FIG. 4 is a schematic illustration of some of the Z blocking groups thatcan be used according to the invention.

FIG. 5 shows two cycles of incorporation of labelled and blocked DGTP,DCTP and dATP respectively (compounds 18, 24 and 32).

FIG. 6 shows six cycles of incorporation of labelled and blocked DTTP(compound 6).

FIG. 7 shows the effective blocking by compound 38 (a 3′-Oallylnucleotide of the invention).

The present invention relates to nucleotide or nucleoside molecules thatare modified by the reversible covalent attachment of a 3′-OH blockinggroups thereto, and which molecules may be used in reactions whereblocked nucleotide or nucleoside molecules are required, such as insequencing reactions, polynucleotide synthesis and the like.

Where the blocking group is an allyl group, it may be introduced intothe 3′-position using standard literature procedures such as that usedby Metzker (infra).

The allyl groups are removed by reacting in aqueous solution a compoundof formula R—O-allyl, R₂N(allyl), RNH(allyl), RN(allyl)₂ or R—S-allyl(wherein R is a water-soluble biological molecule) with a transitionmetal, wherein said transition metal is capable of forming a metal allylcomplex, in the presence of one or more ligands selected from the groupcomprising water-soluble phosphine and water-soluble mixednitrogen-phosphine ligands.

The water-soluble biological molecule is not particularly restrictedprovided, of course, it contains one or more hydroxyl, acid, amino,amide or thiol functionalities protected with an allyl group. Allylesters are examples of compounds of formula R—O-allyl. Preferredfunctionalities are hydroxyl and amino.

As used herein the term biological molecule is used to embrace anymolecules or class of Molecule which performs a biological role. Suchmolecules include for example, polynucleotides such as DNA and RNA,oligonucleotides and single nucleotides. In addition, peptides andpeptide mimetics, such as enzymes and hormones etc., are embraced by theinvention. Compounds which comprise a secondary amide linkage, such aspeptides, or a secondary amine, where such compounds are allylated onthe nitrogen atom of the secondary amine or amide, are examples ofcompounds of formula R₂N(allyl) in which both R groups belong to thesame biological molecule. Particularly preferred compounds however arepolynucleotides, (including oligonucleotides) and nucleotides andnucleosides, preferably those which contain one base to which isattached a detectable label linked through a cleavable linker. Suchcompounds are useful in the determination of sequences ofoligonucleotides as described herein.

Transition metals of use in the invention are any which may form metalallyl complexes, for example platinum, palladium, rhodium, ruthenium,osmium and iridium. Palladium is preferred.

The transition metal, e.g. palladium, is conveniently introduced as asalt, e.g. as a halide. Mixed salts such as Na₂PdCl₄ may also be used.Other appropriate salts and compounds will be readily determined by theskilled person and are commercially available, e.g. from AldrichChemical Company.

Suitable ligands are any phosphine or mixed nitrogen-phosphine ligandsknown to those skilled in the art, characterised in that the ligands arederivatised so as to render them water-soluble, e.g. by introducing oneor more sulfonate, amine, hydroxyl (preferably a plurality of hydroxyl)or carboxylate residues. Where amine residues are present, formation ofamine salts may assist the solublisation of the ligand and thus themetal-allyl complex. Examples of appropriate ligands are triarylphosphines, e.g. triphenyl phosphine, derivatised so as to make themwater-soluble. Also preferred are trialkyl phosphines, e.g.tri-C₁₋₆-alkyl phosphines such as triethyl phosphines; such trialkylphosphines are likewise derivatised so as to make them water-soluble.Sulfonate-containing and carboxylate-containing phosphines areparticularly preferred; an example of the former3,3′,3″-phosphinidynetris (benzenesulfonic acid) which is commerciallyavailable from Aldrich Chemical Company as the trisodium salt; and apreferred example of the latter is tris(2-carboxyethyl)phosphine whichis available from Aldrich as the hydrochloride salt.

The derivatised water-soluble phosphines and nitrogen-containingphosphines described herein may be used as their salts (e.g. as thehydrochloride or sodium salts) or, for example, in the case of thesulfonic and carboxylic acid-containing phosphines described herein, asthe free acids. Thus 3,3′,3″-phosphinidynetris (benzenesulfonic acid)and tris(2-carboxyethyl)phosphines may be introduced either as thetriacids or the trisodium salts. Other appropriate salts will be evidentto those skilled in the art. The existence in salt form is notparticularly important provided the phosphines are soluble in aqueoussolution.

Other ligands which may be used to include the following:

The skilled person will be aware that the atoms chelated to thetransition metal in the water soluble complex may be part of mono- orpolydentate ligands. Some such polydentate ligands are shown above.Whilst monodentate ligands are preferred, the invention thus alsoembraces methods which use water-soluble bi-, tri-, tetra-, penta- andhexadentate water-soluble phosphine and water-solublenitrogen-containing phosphine ligands

The various aspects of the invention relating to allyl blocking groupsare of particular utility in sequencing polynucleotides wherein the3′-OH is allylated. However, when present, the 2′-OH is equally amenableto allylation, and to deprotection according to the method of theinvention if necessary. In fact any allylated alcohol may be deprotectedaccording to the method of the invention. Preferred allylated alcohols,however, are those derived from primary and secondary alcohols.Particularly preferred are allylated nucleosides and nucleotides asdescribed herein. It is possible to deprotect tertiary allylatedalcohols—the reaction is simply slower (although deprotection may be insuch, and other deprotections of this invention, accelerated ifnecessary by heating the solution, e.g. to 40° C., preferably 50° C. orhigher such as approximately 60° C. or even up to 80° C.).

It is also possible to deprotect allylated primary or secondary aminesand allylated thiols.

As noted earlier, the aqueous solution in which allyl deprotection iseffected need not be 100% (as the continuous phase). However,substantially pure water (e.g. at least 98 vol % preferably about 100vol %) is preferred. Cosolvents are generally not required although theycan assist in the solublisation of the allylated substrate for thedeallylation. Generally, biomolecules are readily soluble in water (e.g.pure water) in which the deprotection reaction described herein may beeffected. If desirable, one or more water-miscible cosolvents may beemployed. Appropriate solvents include acetonitrile ordimethylsulfoxide, methanol, ethanol and acetone, methanol beingpreferred. Less preferred solvents include tetrahydrofuran (THF) anddioxane.

In the method of allyl deprotection according to the invention, asoluble metal complex is formed comprising a transition metal and one ormore water-soluble phosphine and water-soluble nitrogen-containingphosphine ligands. More than one type of water-solublephosphine/nitrogen-containing phosphine ligand may be used in adeallylation reaction although generally only one type of these classesof ligand will be used in a given reaction. We believe the deallylationreaction to be catalytic. Accordingly, the quantity of transition metal,e.g. palladium, may be less than 1 mol % (calculated relative to theallyl-protected compound to be deprotected). Advantageously the amountof catalyst may be much less than 1 mol %, e.g. <0.50 mol %, preferably<0.10 mol %, particularly <0.05 mol %. Even lower quantities of metalmay be used, for example <0.03 or even <0.01 mol %. As those skilled inthe art will be aware, however, as quantity of catalyst ds reduced, sotoo is the speed of the reaction. The skilled person will be able tojudge, in any instance, the precise quantity of transition metal andthus catalyst most optimally suited to any particular deallylationreaction.

In contrast to the amount of metal required in forming the activecatalyst, the quantity of water-soluble phosphorus-containing ligand(s)used must be greater than 1 molar equivalent (again calculated relativeto the allyl-protected compound to be deprotected). Preferably greaterthan 4, e.g. greater than 6, for example 8-12 molar equivalents ofligand may be used. Even higher quantities of ligand e.g. >20 moleequivalents may be used if desired.

The skilled person will be able to determine the quantity of ligand bestsuited to any individual reaction.

Where the blocking group is any of —C(R′)₂—O—R″, —C(R′)₂—N(R″)₂,—C(R′)₂—N(H)R″, —C(R′)₂—S—R″ and —C(R′)₂—F, i.e. of formula Z, each R′may be independently H or an alkyl

The intermediates produced advantageously spontaneously dissociate underaqueous conditions back to the natural 3′ hydroxy structure, whichpermits further incorporation of another nucleotide. Any appropriateprotecting group may be used, as discussed herein. Preferably, Z is offormula —C(R′)₂—O—R″, —C(R′)₂—N(R″)₂, —C(R′)₂—N(H)R″ and —C(R′)₂—SR″.Particularly preferably, Z is of the formula —C(R′)₂—O—R″,—C(R′)₂—N(R″)₂, and —C(R′)₂—SR″. R″ may be a benzyl group or asubstituted benzyl group.

One example of groups of structure —O—Z wherein Z is —C(R′)₂—N(R″)₂ arethose in which —N(R″)₂ is azido (—N₃). One preferred such example isazidomethyl wherein each R′ is H. Alternatively, R′ in Z groups offormula —C(R′)₂—N₃ and other Z groups may be any of the other groupsdiscussed herein.

Examples of typical R′ groups include C₁₋₆ alkyl, particularly methyland ethyl, and the following (in which each structure shows the bondwhich connects the R′ moiety to the carbon atom to which it is attachedin the Z groups; the asterisks (*) indicate the points of attachment):

(wherein each R is an optionally substituted C₁₋₁₀ alkyl group, anoptionally substituted alkoxy group, a halogen atom or functional groupsuch as hydroxyl, amino, cyano, nitro, carboxyl and the like) and “Het”is a heterocyclic (which may for example be a heteroaryl group). TheseR′ groups shown above are preferred where the other R′ group is the sameas the first or is hydrogen. Preferred Z groups are of formula C(R′)₂N₃in which the R′ groups are selected from the structures given above andhydrogen; or in which (R′)₂ represents an alkylidene group of formula═C(R′″)₂, e.g. ═C(Me)₂.

Where molecules contain Z groups of formula C(R′)₂N₃, the azido groupmay be converted to amino by contacting such molecules with thephosphine or nitrogen-containing phosphines ligands described in detailin connection with the transition metal complexes which serve to cleavethe allyl groups from compounds of formula PN—O-allyl, formulaR—O-allyl, R₂N(allyl), RNH (allyl), RN(allyl)₂ and R—S-allyl. Whentransforming azido to amino, however, no transition metal is necessary.Alternatively, the azido group in Z groups of formula C(R′)₂N₃ may beconverted to amino by contacting such molecules with the thiols, inparticular water-soluble thiols such as dithiothreitol (DTT).

Where an R′ group represents a detectable label attached through alinking group, the other R′ group or any other part of “Z” willgenerally not contain a detectable label, nor will the base of thenucleoside or nucleotide contain a detectable label. Appropriate linkinggroups for connecting the detectable label to the 3′blocking group willbe known to the skilled person and examples of such groups are describedin greater detail hereinafter.

Exemplary of linkages in R′ groups containing detectable labels arethose which contain one or more amide bonds. Such linkers may alsocontain an arylene, e.g. phenylene, group in the chain (i.e. a linkingmoiety —Ar— where the phenyl ring is part of the linker by way of its1,4-disposed carbon atoms). The phenyl ring may be substituted at itsnon-bonded position with one or more substituents such as alkyl,hydroxyl, alkyloxy, halide, nitro, carboxyl or cyano and the like,particularly electron-withdrawing groups, which electron-withdrawing iseither by induction or resonance. The linkage in the R′ group may alsoinclude moieties such a —O—, —S(O)_(q), wherein q is 0, 1 or 2 or NH orNalkyl. Examples of such Z groups are as follows:

(wherein EWG stands for electron-withdrawing group; n is an integer offrom 1 to 50, preferably 2-20, e.g. 3 to 10; and fluor indicates afluorophore). An example of an electron-withdrawing group by resonanceis nitro; a group which acts through induction is fluoro. The skilledperson will be aware of other appropriate electron-withdrawing groups.In addition, it will be understood that whilst a fluorophore isindicated as being the detectable label present, other detectable groupsas discussed in greater detail hereinafter may be included instead.

Where a detectable label is attached to a nucleotide at the 3′-blockingposition, the linker need not be cleavable to have utility in thosereactions, such as DNA sequencing, described herein which require thelabel to be “read” and removed before the next step of the reaction.This is because the label, when attached to the 3′block, will becomeseparated from the nucleotide when the intermediate compounds describedherein collapse so as to replace the “Z” group with a hydrogen atom. Asnoted above, each R″ is or is part of a removable protecting group. R″may be a benzyl group or is substituted benzyl group is an alternativeembodiment.

It will be appreciated that where it is possible to incorporate adetectable label onto a group R″, the invention embraces thispossibility. Thus, where R″ is a benzyl group, the phenyl ring may beara linker group to which is attached a fluorophore or other detectablegroup. Introduction of such groups does not prevent the ability toremove such R″s and they do not prevent the generation of the desiredunstable intermediates during deprotection of blocking groups of formulaZ.

As is known in the art, a “nucleotide” consists of a nitrogenous base, asugar, and one or more phosphate groups. They are monomeric units of anucleic acid sequence. In RNA, the sugar is a ribose, and in DNA adeoxyribose, i.e. a sugar lacking a hydroxyl group that is present inribose. The nitrogenous base is a derivative of purine or pyrimidine.The purines are adenine (A) and guanine (G), and the pyrimidines arecytosine (C) and thymine (T) (or in the context of RNA, uracil (U)). TheC-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of apurine. A nucleotide is also a phosphate ester or a nucleoside, withesterification occurring on the hydroxyl group attached to C-5 of thesugar. Nucleotides are usually mono, di- or triphosphates.

A “nucleoside” is structurally similar to a nucleotide, but is missingthe phosphate moieties. An example of a nucleoside analogue would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, theskilled person will appreciate that derivatives and analogues areavailable which do not alter the capability of the nucleotide ornucleoside to undergo Watson-Crick base pairing. “Derivative” or“analogue” means a compound or molecule whose core structure is the sameas, or closely resembles that of, a parent compound, but which has achemical or physical modification, such as a different or additionalside group, or 2′ and or 3′ blocking groups, which allows the derivativenucleotide or nucleoside to be linked to another molecule. For example,the base can be a deazapurine. The derivatives should be capable ofundergoing Watson-Crick pairing. “Derivative” and “analogue” also mean asynthetic nucleotide or nucleoside derivative having modified basemoieties and/or modified sugar moieties. Such derivatives and analogsare discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son,1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotideanalogs can also comprise modified phosphodiester linkages, includingphosphorothioate, phosphorodithioate, alkyl-phosphonate,phosphoranilidate and phosphoramidate linkages. The analogs should becapable of undergoing Watson-Crick base pairing. “Derivative”, “analog”and “modified” as used herein, may be used interchangeably, and areencompassed by the terms “nucleotide” and “nucleoside” defined herein.

In the context of the present invention, the term “incorporating” meansbecoming part of a nucleic acid (eg DNA) molecule or oligonucleotide orprimer. An oligonucleotide refers to a synthetic or natural moleculecomprising a covalently linked sequence of nucleotides which are formedby a phosphodiester or modified phosphodiester bond between the 3′position of the pentose on one nucleotide and the 5′ position of thepentose on an adjacent nucleotide.

The term “alkyl” covers straight chain, branched chain and cycloalkylgroups. Unless the context indicates otherwise, the term “alkyl” refersto groups having 1 to 10 carbon atoms, for example 1 to 8 carbon atoms,and typically from 1 to 6 carbon atoms, for example from 1 to 4 carbonatoms. Examples of alkyl groups include methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl,2-methyl butyl, 3-methyl butyl, and n-hexyl and its isomers.

Examples of cycloalkyl groups are those having from 3 to 10 ring atoms,particular examples including those derived from cyclopropane,cyclobutane, cyclopentane, cyclohexane and cycloheptane, bicycloheptaneand decalin.

Where alkyl (including cycloalkyl) groups are substituted, particularlywhere these form either both of the R′ groups of the molecules of theinvention, examples of appropriate substituents include halogensubstituents or functional groups such as hydroxyl, amino, cyano, nitro,carboxyl and the like. Such groups may also be substituents, whereappropriate, of the other R′ groups in the molecules of the invention.

The term amino refers to groups of type NR*R**, wherein R* and R** areindependently selected from hydrogen, a C₁₋₆ alkyl group (also referredto as C₁₋₆ alkylamino or di-C₁₋₆ alkylamino).

The term “halogen” as used herein includes fluorine, chlorine, bromineand iodine.

The nucleotide molecules of the present invention are suitable for usein many different methods where the detection of nucleotides isrequired.

DNA sequencing methods, such as those outlined in U.S. Pat. No.5,302,509 can be carried out using the nucleotides.

The present invention can make use of conventional detectable labels.Detection can be carried out by any suitable method, includingfluorescence spectroscopy or by other optical means. The preferred labelis a fluorophore, which, after absorption of energy, emits radiation ata defined wavelength. Many suitable fluorescent labels are known. Forexample, Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) disclosesdansyl-functionalised fluorescent moieties that can be used in thepresent invention. Zhu et al. (Cytometry 28:206-211, 1997) describes theuse of the fluorescent labels Cy3 and Cy5, which can also be used in thepresent invention. Labels suitable for use are also disclosed in Proberet al. (Science 238:336-341, 1987); Connell et al. (BioTechniques5(4):342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15(11):4593-4602,1987) and Smith et al. (Nature 321:674, 1986). Other commerciallyavailable fluorescent labels include, but are not limited to,fluorescein, rhodamine (including TMR, texas red and Rox), alexa,bodipy, acridine, coumarin, pyrene, benzanthracene and the cyanins.

Multiple labels can also be used in the invention. For example,bi-fluorophore FRET cassettes (Tet. Let. 46:8867-8871, 2000) are wellknown in the art and can be utilised in the present invention.Multi-fluor dendrimeric systems (J. Amer. Chem. Soc. 123:8101-8108,2001) can also be used.

Although fluorescent labels are preferred, other forms of detectablelabels will be apparent as useful to those of ordinary skill. Forexample, microparticles, including quantum dots (Empodocles et al.,Nature 399:126-130, 1999), gold nanoparticles (Reichert et al., Anal.Chem. 72:6025-6029, 2000) and microbeads (Lacoste et al., Proc. Natl.Acad. Sci USA 97(17):9461-9466, 2000) can all be used.

Multi-component labels can also be used in the invention. Amulti-component label is one which is dependent on the interaction witha further compound for detection. The most common multi-component labelused in biology is the biotin-streptavidin system. Biotin is used as thelabel attached to the nucleotide base. Streptavidin is then addedseparately to enable detection to occur. Other multi-component systemsare available. For example, dinitrophenol has a commercially availablefluorescent antibody that can be used for detection.

The invention has been and will be further described with reference tonucleotides. However, unless indicated otherwise, the reference tonucleotides is also intended to be applicable to nucleosides. Theinvention will also be further described with reference to DNA, althoughthe description will also be applicable to RNA, PNA, and other nucleicacids, unless otherwise indicated.

The modified nucleotides of the invention may use a cleavable linker toattach the label to the nucleotide. The use of a cleavable linkerensures that the label can, if required, be removed after detection,avoiding any interfering signal with any labelled nucleotideincorporated subsequently.

Generally, the use of cleavable linkers is preferable, particularly inthe methods of the invention hereinbefore described except where thedetectable label is attached to the nucleotide by forming part of the“Z” group.

Those skilled in the art will be aware of the utility ofdideoxynucleoside triphosphates in so-called Sanger sequencing methods,and related protocols (Sanger-type), which rely upon randomisedchain-termination at a particular type of nucleotide. An example of aSanger-type sequencing protocol is the BASS method described by Metzker(infra). Other Sanger-type sequencing methods will be known to thoseskilled in the art.

Sanger and Sanger-type methods generally operate by the conducting of anexperiment in which eight types of nucleotides are provided, four ofwhich contain a 3′OH group; and four of which omit the OH group andwhich are labeled differently from each other. The nucleotides usedwhich omit the 3′OH group—dideoxy nucleotides—are conventiallyabbreviated to ddNTPs. As is known by the skilled person, since theddNTPs are labeled differently, by determining the positions of theterminal nucleotides incorporated, and combining this information, thesequence of the target oligonucleotide may be determined.

The nucleotides of the present invention, it will be recognized, may beof utility in Sanger methods and related protocols since the same effectachieved by using ddNTPs may be achieved by using the novel 3′-OHblocking groups described herein: both prevent incorporation ofsubsequent nucleotides.

The use of the nucleotides according to the present invention in Sangerand Sanger-type sequencing methods, wherein the linker connecting thedetectable label to the nucleotide may or may not be cleavable, forms astill further aspect of this invention. Viewed from this aspect, theinvention provides the use of such nucleotides in a Sanger or aSanger-type sequencing method.

Where 3′-OH Z-blocked nucleotides according to the present invention areused, it will be appreciated that the detectable labels attached to thenucleotides need not be connected via cleavable linkers, since in eachinstance where a labelled nucleotide of the invention is incorporated,no nucleotides need to be subsequently incorporated and thus the labelneed not be removed from the nucleotide.

Moreover, it will be appreciated that monitoring of the incorporation of3′OH blocked nucleotides may be determined by use of radioactive ³²P inthe phosphate groups attached. These may be present in either the ddNTPsthemselves or in the primers used for extension. Where the blockinggroups are of formula “Z”, this represents a further aspect of theinvention.

Viewed from this aspect, the invention provides the use of a nucleotidehaving a 3′OH group blocked with a “Z” group in a Sanger or aSanger-type sequencing method. In this embodiment, a ³²P detectablelabel may be present in either the ddNTPs used in the primer used forextension.

Cleavable linkers are known in the art, and conventional chemistry canbe applied to attach a linker to a nucleotide base and a label. Thelinker can be cleaved by any suitable method, including exposure toacids, bases, nucleophiles, electrophiles, radicals, metals, reducing oroxidising agents, light, temperature, enzymes etc. The linker asdiscussed herein may also be cleaved with the same catalyst used tocleave the 3′O-blocking group bond. Suitable linkers can be adapted fromstandard chemical blocking groups, as disclosed in Greene & Wuts,Protective Groups in Organic Synthesis, John Wiley & Sons. Furthersuitable cleavable linkers used in solid-phase synthesis are disclosedin Guillier et al. (Chem. Rev. 100:2092-2157, 2000).

The use of the term “cleavable linker” is not meant to imply that thewhole linker is required to be removed from e.g., the nucleotide base.Where the detectable label is attached to the base, the nucleosidecleavage site can be located at a position on the linker that ensuresthat part of the linker remains attached to the nucleotide base aftercleavage.

Where the detectable label is attached to the base, the linker can beattached at any position on the nucleotide base provided thatWatson-Crick base pairing can still be carried out. In the context ofpurine bases, it is preferred if the linker is attached via the7-position of the purine or the preferred deazapurine analogue, via an8-modified purine, via an N-6 modified adenosine or an N-2 modifiedguanine. For pyrimidines, attachment is preferably via the 5-position oncytosine, thymidine or uracil and the N-4 position on cytosine. Suitablenucleotide structures are shown in FIG. 1. For each structure in FIG. 1X can be H, phosphate, diphosphate or triphosphate. R₁ and R₂ can be thesame or different, and are selected from H, OH, O-allyl, or formula Z asdescribed herein or any other group which can be transformed into an OH,including, but not limited to, a carbonyl, provided that at least one ofR₁ and R₂ is O-allyl or formula Z as described herein. Some suitablefunctional groups for R₁ and R₂ include the structures shown in FIGS. 3and 4.

Suitable linkers are shown in FIG. 3 and include, but are not limitedto, disulfide linkers (1), acid labile linkers (2, 3, 4 and 5; includingdialkoxybenzyl linkers (e.g., 2), Sieber linkers (e.g., 3), indolelinkers (e.g., 4), t-butyl Sieber linkers (e.g., 5)), electrophilicallycleavable linkers, nucleophilically cleavable linkers, photocleavablelinkers, cleavage under reductive conditions, oxidative conditions,cleavage via use of safety-catch linkers, and cleavage by eliminationmechanisms.

A. Electrophilically Cleaved Linkers.

Electrophilically cleaved linkers are typically cleaved by protons andinclude cleavages sensitive to acids. Suitable linkers include themodified benzylic systems such as trityl, p-alkoxybenzyl esters andp-alkoxybenzyl amides. Other suitable linkers includetert-butyloxycarbonyl (Boc) groups and the acetal system.

The use of thiophilic metals, such as nickel, silver or mercury, in thecleavage of thioacetal or other sulfur-containing protecting groups canalso be considered for the preparation of suitable linker molecules.

B. Nucleophilically Cleaved Linkers.

Nucleophilic cleavage is also a well recognised method in thepreparation of linker molecules. Groups such as esters that are labilein water (i.e., can be cleaved simply at basic pH) and groups that arelabile to non-aqueous nucleophiles, can be used. Fluoride ions can beused to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or t-butyldimethyl silane (TBDMS).

C. Photocleavable Linkers.

Photocleavable linkers have been used widely in carbohydrate chemistry.It is preferable that the light required to activate cleavage does notaffect the other components of the modified nucleotides. For example, ifa fluorophore is used as the label, it is preferable if this absorbslight of a different wavelength to that required to cleave the linkermolecule. Suitable linkers include those based on O-nitrobenzylcompounds and nitroveratryl compounds. Linkers based on benzoinchemistry can also be used (Lee et al., J. Org. Chem. 64:3454-3460,1999).

D. Cleavage Under Reductive Conditions

There are many linkers known that are susceptible to reductive cleavage.Catalytic hydrogenation using palladium-based catalysts has been used tocleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction isalso known in the art.

E. Cleavage Under Oxidative Conditions

Oxidation-based approaches are well known in the art. These includeoxidation of p-alkoxybenzyl groups and the oxidation of sulfur andselenium linkers. The use of aqueous iodine to cleave disulfides andother sulfur or selenium-based linkers is also within the scope of theinvention.

F. Safety-Catch Linkers

Safety-catch linkers are those that cleave in two steps. In a preferredsystem the first step is the generation of a reactive nucleophiliccenter followed by a second step involving an intra-molecularcyclization that results in cleavage. For example, levulinic esterlinkages can be treated with hydrazine or photochemistry to release anactive amine, which can then be cyclised to cleave an ester elsewhere inthe molecule (Burgess et al., J. Org. Chem. 62:5165-5168, 1997).

G. Cleavage by Elimination Mechanisms

Elimination reactions can also be used. For example, the base-catalysedelimination of groups such as Fmoc and cyanoethyl, andpalladium-catalysed reductive elimination of allylic systems, can beused.

As well as the cleavage site, the linker can comprise a spacer unit. Thespacer distances e.g., the nucleotide base from the cleavage site orlabel. The length of the linker is unimportant provided that the labelis held a sufficient distance from the nucleotide so as not to interferewith any interaction between the nucleotide and an enzyme:

In a preferred embodiment the linker may consist of the samefunctionality as the block. This will make the deprotection anddeblocking process more efficient, as only a single treatment will berequired to remove both the label and the block.

Particularly preferred linkers are phosphine-cleavable azide containinglinkers.

A method for determining the sequence of a target polynucleotide can becarried out by contacting the target polynucleotide separately with thedifferent nucleotides to form the complement to that of the targetpolynucleotide, and detecting the incorporation of the nucleotides. Sucha method makes use of polymerisation, whereby a polymerase enzymeextends the complementary strand by incorporating the correct nucleotidecomplementary to that on the target. The polymerisation reaction alsorequires a specific primer to initiate polymerisation.

For each cycle, the incorporation of the modified nucleotide is carriedout by the polymerase enzyme, and the incorporation event is thendetermined. Many different polymerase enzymes exist, and it will beevident to the person of ordinary skill which is most appropriate touse. Preferred enzymes include DNA polymerase I, the Klenow fragment,DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase or Ventpolymerase. Polymerases engineered to have specific properties can alsobe used. As noted earlier, the molecule is preferably incorporated by apolymerase and particularly from Thermococcus sp., such as 9° N. Evenmore preferably, the polymerase is a mutant 9° N A485L and even morepreferably is a double mutant Y409V and A485L. An example of one suchpreferred enzyme is Thermococcus sp. 9° N exo −Y409V A485L availablefrom New England Biolabs. Examples of such appropriate polymerases aredisclosed in Proc. Natl. Acad. Sci. USA, 1996(93), pp 5281-5285, NucleicAcids Research, 1999(27), pp 2454-2553 and Acids Research, 2002(30), pp605-613.

The sequencing methods are preferably carried out with the targetpolynucleotide arrayed on a solid support. Multiple targetpolynucleotides can be immobilised on the solid support through linkermolecules, or can be attached to particles, e.g., microspheres, whichcan also be attached to a solid support material. The polynucleotidescan be attached to the solid support by a number of means, including theuse of biotin-avidin interactions. Methods for immobilizingpolynucleotides on a solid support are well known in the art, andinclude lithographic techniques and “spotting” individualpolynucleotides in defined positions on a solid support. Suitable solidsupports are known in the art, and include glass slides and beads,ceramic and silicon surfaces and plastic materials. The support isusually a flat surface although microscopic beads (microspheres) canalso be used and can in turn be attached to another solid support byknown means. The microspheres can be of any suitable size, typically inthe range of from 10 nm to 100 nm in diameter. In a preferredembodiment, the polynucleotides are attached directly onto a planarsurface, preferably a planar glass surface. Attachment will preferablybe by means of a covalent linkage. Preferably, the arrays that are usedare single molecule arrays that comprise polynucleotides in distinctoptically resolvable areas, e.g., as disclosed in InternationalApplication No. WO00/06770.

The sequencing method can be carried out on both single polynucleotidemolecule and multi-polynucleotide molecule arrays, i.e., arrays ofdistinct individual polynucleotide molecules and arrays of distinctregions comprising multiple copies of one individual polynucleotidemolecule. Single molecule arrays allow each individual polynucleotide tobe resolved separately. The use of single molecule arrays is preferred.Sequencing single molecule arrays non-destructively allows a spatiallyaddressable array to be formed.

The method makes use of the polymerisation reaction to generate thecomplementary sequence of the target. Conditions compatible withpolymerization reactions will be apparent to the skilled person.

To carry out the polymerase reaction it will usually be necessary tofirst anneal a primer sequence to the target polynucleotide, the primersequence being recognised by the polymerase enzyme and acting as aninitiation site for the subsequent extension of the complementarystrand. The primer sequence may be added as a separate component withrespect to the target polynucleotide. Alternatively, the primer and thetarget polynucleotide may each be part of one single stranded molecule,with the primer portion forming an intramolecular duplex with a part ofthe target, i.e., a hairpin loop structure. This structure may beimmobilised to the solid support at any point on the molecule. Otherconditions necessary for carrying out the polymerase reaction, includingtemperature, pH, buffer compositions etc., will be apparent to thoseskilled in the art.

The modified nucleotides of the invention are then brought into contactwith the target polynucleotide, to allow polymerisation to occur. Thenucleotides may be added sequentially, i.e., separate addition of eachnucleotide type (A, T, G or C), or added together. If they are addedtogether, it is preferable for each nucleotide type to be labelled witha different label.

This polymerisation step is allowed to proceed for a time sufficient toallow incorporation of a nucleotide.

Nucleotides that are not incorporated are then removed, for example, bysubjecting the array to a washing step, and detection of theincorporated labels may then be carried out.

Detection may be by conventional means, for example if the label is afluorescent moiety, detection of an incorporated base may be carried outby using a confocal scanning microscope to scan the surface of the arraywith a laser, to image a fluorophore bound directly to the incorporatedbase. Alternatively, a sensitive 2-D detector, such as a charge-coupleddetector (CCD), can be used to visualise the individual signalsgenerated. However, other techniques such as scanning near-field opticalmicroscopy (SNOM) are available and may be used when imaging densearrays. For example, using SNOM, individual polynucleotides may bedistinguished when separated by a distance of less than 100 nm, e.g., 10nm to 10 μm. For a description of scanning near-field opticalmicroscopy, see Moyer et al., Laser Focus World 29:10, 1993. Suitableapparatus used for imaging polynucleotide arrays are known and thetechnical set-up will be apparent to the skilled person.

After detection, the label may be removed using suitable conditions thatcleave the linker and the 3′OH block to allow for incorporation offurther modified nucleotides of the invention. Appropriate conditionsmay be those described herein for allyl group and for “Z” groupdeprotections. These conditions can serve to deprotect both the linker(if cleavable) and the blocking group. Alternatively, the linker may bedeprotected separately from the allyl group by employing methods ofcleaving the linker known in the art (which do not sever the 0-blockinggroup bond) followed by deprotection.

This invention may be further understood with reference to the followingexamples which serve to illustrate the invention and not to limit itsscope. 3′-OH protected with an azidomethyl group as a protected form ofa hemiaminal:

Nucleotides bearing this blocking group at the 3′position have beensynthesised, shown to be successfully incorporated by DNA polymerases,block efficiently and may be subsequently removed under neutral, aqueousconditions using water soluble phosphines or thiols allowing furtherextension:

5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyuridine (1)

To a solution of 5-iodo-2′-deoxyuridine (1.05 g, 2.96 mmol) and CuI (114mg, 0.60 mmol) in dry DMF (21 ml) was added triethylamine (0.9 ml).After stirring for 5 min trifluoro-N-prop-2-ynyl-acetamide (1.35 g, 9.0mmol) and Pd(PPh₃)₄ (330 mg, 0.29 mmol) were added to the mixture andthe reaction was stirred at room temperature in the dark for 16 h.Metanol (MeOH) (40 ml) and bicarbonate dowex added to the reactionmixture and stirred for 45 min. The mixture was filtered and thefiltrate washed with MeOH and the solvent was removed under vacuum. Thecrude mixture was purified by chromatography on silica (ethyl acetate(EtOAc) to EtOAc:MeOH 95:5) to give slightly yellow crystals (794 mg,71%). ¹H NMR (d₆ dimethylsulfoxide (DMSO)) δ 2.13-2.17 (m, 2H, H-2′),3.57-3.65 (m, 2H, H-5′), 3.81-3.84 (m, 1H, H-4′), 4.23-4.27 (m, 3H,H-3′, CH₂N), 5.13 (t, J=5.0 Hz, 1H, OH), 5.20 (d, J=4.3 Hz, 1H, OH),6.13 (t, J=6.7 Hz, 1H, H-1′), 8.23 (s, 1H, H-6), 10.11 (t, J=5.6 Hz, 1H,NH), 11.70 (br s, 1H, NH). Mass (−ve electrospray) calcd forC₁₄H₁₄F₃N₃O₆ 377.08, found 376.

5′-O-(tert-butydimethylsilyl)-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyuridine(2)

To a solution of (1) (656 mg, 1.74 mmol) in dry DMF (15 ml) was addedt-butyldimethylsilylchloride (288 mg, 1.91 mmol) in small portions,followed by imidazole (130 mg, 1.91 mmol). The reaction was followed byTLC and was completed after stirring for 8 h at room temperature. Thereaction was quenched with sat. aq. NaCl solution. EtOAc (25 ml) wasadded to the reaction mixture and the aqueous layer was extracted withEtOAc three times. After drying the combined organics (MgSO₄), thesolvent was removed under vacuum. Purification by chromatography onsilica (EtOAc:petroleum ether 8:2) gave (2) as slightly yellow crystals(676 mg, 83%). ¹H NMR (d₆ DMSO) δ 0.00 (s, 6H, CH₃), 0.79 (s, 9H, tBu),1.93-2.00 (m, 1H, H-2′), 2.06-2.11 (m, 1H, H-2′), 3.63-3.75 (m, 2H,H-5′), 3.79-3.80 (m, 1H, H-4′), 4.12-4.14 (m, 3H, H-3′, CH₂N), 5.22 (d,J=4.1 Hz, 1H, OH), 6.03 (t, J=6.9 Hz, 1H, H-1′), 7.86 (s, 1H, H-6), 9.95(t, J=5.4 Hz, 1H, NH), 11.61 (br s, 1H, NH). Mass (−ve electrospray)calcd for C₂₀H₂₈F₃N₃O₆Si 491.17, found 490.

5′-O-(tert-Butydimethylsilyl)-3′-O-methylthiomethyl-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyuridine(3)

To a solution of (2) (1.84 g, 3.7 mmol) in dry DMSO (7 ml) was addedacetic acid (3.2 ml) and acetic anhydride (10.2 ml). The mixture wasstirred for 2 days at room temperature, before it was quenched with sat.aq. NaHCO₃. EtOAc (50 ml) was added and the aqueous layer was extractedthree times with ethyl acetate. The combined organic layers were washedwith sat. aq. NaHCO₃ solution and dried (MgSO₄). After removing thesolvent under reduced pressure, the product (3) was purified bychromatography on silica (EtOAc:petroleum ether 8:2) yielding a clearsticky oil (1.83 g, 89%). ¹H NMR (d₆ DMSO): δ 0.00 (s, 6H, CH₃), 0.79(s, 9H, tBu), 1.96-2.06 (m, 1H, H-2′), 1.99 (s, 3H, SCH₃), 2.20-2.26 (m,1H, H-2′), 3.63-3.74 (m, 2H, H-5′), 3.92-3.95 (m, 1H, H-4′), 4.11-4.13(m, 2H, CH₂), 4.28-4.30 (m, 1H, H-3′), 4.59 (br s, 2H, CH₂), 5.97 (t,J=6.9 Hz, 1H, H-1′), 7.85 (s, 1H, H-6), 9.95 (t, J=5.3 Hz, 1H, NH),11.64 (s, 1H, NH). Mass (−ve electrospray) calcd for C₂₂H₃₂F₃N₃O₆SSi551.17, found 550.

3′-O-Azidomethyl-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyuridine(4)

To a solution of (3) (348 mg, 0.63 mmol) and cyclohexene (0.32 ml, 3.2mmol) in dry CH₂Cl₂ (5 ml) at 4° C., sulfurylchoride (1M in CH₂Cl₂, 0.76ml, 0.76 mmol) was added drop wise under N₂. After 10 min TLC indicatedthe full consumption of the nucleoside (3). The solvent was evaporatedand the residue was subjected to high vacuum for 20 min. It was thenredissolved in dry DMF (3 ml) and treated with NaN₃ (205 mg, 3.15 mmol).The resulting suspension was stirred under room temperature for 2 h. Thereaction was quenched with CH₂Cl₂ and the organic layers were washedwith sat aq. NaCl solution. After removing the solvent, the resultingyellow gum was redissolved in THF (2 ml) and treated with TBAF (1 M inTHF, 0.5 ml) at room temperature for 30 min. The solvent was removed andthe reaction worked up with CH₂Cl₂ and sat. aq. NaHCO₃ solution. Theaqueous layer was extracted three times with CH₂Cl₂. Purification bychromatography on silica (EtOAc:petroleum ether 1:1 to EtOAc) gave (4)(100 mg, 37%) as a pale yellow foam. ¹H NMR (d₆ DMSO) δ 2.15-2.26 (m,2H, H-2′), 3.47-3.57 (m, 2H, H-5′), 3.88-3.90 (m, 1H, H-4′), 4.14 (d,J=4.7 Hz, 2H, CH₂NH), 4.24-4.27 (m, 1H, H-3′), 4.75 (s, 2H, CH₂N₃), 5.14(t, J=5.2 Hz, 1H, OH), 5.96-6.00 (m, 1H, H-1′), 8.10 (s, 1H, H-6), 10.00(s, 1H, NHCOCF₃)), 11.26 (s, 1H, NH).

Preparation of bis(tri-n-butylammonium) pyrophosphate (0.5 M solution inDMF)

Tetrasodium diphosphate decahydrate (1.5 g, 3.4 mmol) was dissolved inwater (34 ml) and the solution was applied to a column of dowex in theH⁺ form. The column was eluted with water. The eluent dropped directlyinto a cooled (ice bath) and stirred solution of tri-n-butylamine (1.6ml, 6.8 mmol) in EtOH (14 ml). The column was washed until the pH of theeluent increased to 6. The aq. ethanol solution was evaporated todryness and then co-evaporated twice with ethanol and twice withanhydrous DMF. The residue was dissolved in DMF (6.7 ml). The paleyellow solution was stored over 4 Å molecular sieves.

3′-O-Azidomethyl-5-(3-amino-prop-1-ynyl)-2′-deoxyuridine 5′-O-nucleosidetriphosphate (5)

The nucleoside (4) and proton sponge was dried over P₂O₅ under vacuumovernight. A solution of (4) (92 mg, 0.21 mmol) and proton sponge (90mg, 0.42 mmol) in trimethylphosphate (0.5 ml) was stirred with 4 Åmolecular sieves for 1 h. Freshly distilled POCl₃ (24 μl, 0.26 mmol) wasadded and the solution was stirred at 4° C. for 2 h. The mixture wasslowly warmed up to room temperature and bis (tri-n-butyl ammonium)pyrophosphate (1.7 ml, 0.85 mmol) and anhydrous tri-n-butyl amine (0.4ml, 1.7 mmol) was added. After 3 min, the reaction was quenched with 0.1M TEAB (triethylammonium bicarbonate) buffer (15 ml) and stirred for 3h. The water was removed under reduced pressure and the resultingresidue dissolved in concentrated ammonia (ρ 0.88, 15 ml) and stirred atroom temperature for 16 h. The reaction mixture was then evaporated todryness. The residue was dissolved in water and the solution applied toa DEAE-Sephadex A-25 column. MPLC was performed with a linear gradientof TEAB. The triphosphate was eluted between 0.7 M and 0.8 M buffer.Fractions containing the product were combined and evaporated todryness. The residue was dissolved in water and further purified byHPLC. HPLC: t_(r)(5): 18.8 min (Zorbax C18 preparative column, gradient:5% to 35% B in 30 min, buffer A 0.1M TEAB, buffer B MeCN) The productwas isolated as a white foam (76 O.D., 7.6 μmol, 3.8%, ε₂₈₀=10000). ¹HNMR (D₂O) δ 1.79 (s, CH₂), 2.23-2.30; 2.44-2.50 (2×m, 2H, H-2′), 3.85(m, CH₂NH), 4.10-4.18 (m, 2H, H-5′), 4.27 (br s, H-4′), 4.48-4.50 (m,H-3′), 4.70-4.77 (m, CH₂N₃), 6.21 (t, J=6.6 Hz, H-1′), 8.32 (s, 1H,H-6). ³¹P NMR (D₂O) δ −6.6 (m, 1P, P_(γ)), −10.3 (d, J=18.4 Hz, 1P,P_(α)), −21.1 (m, 1P, P_(β)). Mass (−ve electrospray) calcd forC₁₃H₁₉N₆O₁₄P₃ 576.02, found 575.

Cy-3Disulfide Linker

The starting disulfide (4.0 mg, 13.1 μmol) was dissolved in DMF (300 μL)and diisopropylethylamine (4 μL) was slowly added. The mixture wasstirred at room temperature and a solution of Cy-3 dye (5 mg, 6.53 μmol)in DMF (300 μL) was added over 10 min. After 3.5 h, on completereaction, the volatiles were evaporated under reduced pressure and thecrude residue was HPLC purified on a Zorbax analytical column SB-C18with a flow rate of 1 ml/min in 0.1M triethylammonium bicarbonate buffer(buffer A) and CH₃CN (buffer B) using the following gradient: 5 min 2%B; 31 min 55% B; 33 min 95% B; 37 min 95%; 39 min 2% B; 44 min. 2% B.The expected Cy3-disulfide linker was eluted with a t_(r): 21.8 min. in70% yield (based on a UV measurement; ε₅₅₀ 150,000 cm⁻¹ M⁻¹ in H₂O) as ahygroscopic solid. ¹H NMR (D₂O) δ 1.31-1.20 (m+t, J=7.2 Hz, 5H,CH₂+CH₃), 1.56-1.47 (m, 2H, CH₂), 1.67 (s, 12H, 4 CH₃), 1.79-1.74 (m,2H, CH₂), 2.11 (t, J=6.9 Hz, 2H, CH₂), 2.37 (t, J=6.9 Hz, 2H, CH₂), 2.60(t, J=6.3 Hz, 2H, CH₂), 2.67 (t, J=6.9 Hz, 2H, CH₂), 3.27 (t, J=6.1 Hz,2H, CH₂), 4.10-4.00 (m, 4H, 2CH₂), 6.29 (dd, J=13.1, 8.1 Hz, 2H, 2 ═CH),7.29 (dd, 2H, J=8.4, 6.1 Hz, 2 ═CH), 7.75-7.71 (m, 2H, 2 ═CH), 7.78 (s,2H, ═CH), 8.42 (t, J=12.8 Hz, 1H, ═CH). Mass (−ve electrospray) calcdfor C₃₆H₄₇N₃O₉S₄ 793.22, found 792 (M−H), 396 [M/2].

A mixture of Cy3 disulphide linker (2.5 μmol), disuccinimidyl carbonate(0.96 mg, 3.75 μmol) and DMAP (0.46 mg, 3.75 μmol) were dissolved in dryDMF (0.5 ml) and stirred at room temperature for 10 min. The reactionwas monitored by TLC (MeOH:CH₂Cl₂ 3:7) until all the dye linker wasconsumed. Then a solution of (5) (7.5 μmol) and n-Bu₃N (30 μl, 125 μmol)in DMF (0.2 ml) was added to the reaction mixture and stirred at roomtemperature for 1 h. TLC (MeOH:CH₂Cl₂ 4:6) showed complete consumptionof the activated ester and a dark red spot appeared on the baseline. Thereaction was quenched with TEAB buffer (0.1M, 10 ml) and loaded on aDEAE Sephadex column (2×5 cm). The column was first eluted with 0.1 MTEAS buffer (100 ml) to wash off organic residues and then 1 M TEABbuffer (100 ml). The desired triphosphate analogue (6) was eluted outwith 1 M TEAB buffer. The fraction containing the product were combined,evaporated and purified by HPLC. HPLC conditions: t_(r)(6): 16.1 min(Zorbax C18 preparative column, gradient: 2% to 55% B in 30 min, bufferA 0.1M TEAB, buffer B MeCN). The product was isolated as dark red solid(1.35 μmol, 54%, ε₅₅₀=150000). ¹H NMR (D₂O) δ 1.17-1.28 (m, 6H 3×CH₂),1.41-1.48 (m, 3H, CH₃), 1.64 (s, 12H, 4×CH₃), 1.68-1.71 (m, 2H, CH₂),2.07-2.10 (m, 3H, H-2′, CH₂), 2.31-2.35 (m, 1H, H-2′), 2.50-2.54 (m, 2H,CH₂), 2.65 (t, =5.9 Hz, 2H, CH₂), 2.76 (t, J=7.0 Hz, 2H, CH₂), 3.26-3.31(m, 2H, CH₂), 3.88-3.91 (m, 2H CH₂), 3.94-4.06 (m, 3H, CH₂N, H-5′), 4.16(br s, 1H, H-4′), 4.42-4.43 (m, 1H, H-3′), 4.72-4.78 (m, 2H, CH₂N₃),6.24 (dd, J=5.8, 8.2 Hz, H-1′), 6.25 (dd, J=3.5, 8.5 Hz, 2H, H_(Ar)),7.24, 7.25 (2d, J=14.8 Hz, 2×═CH), 7.69-7.86 (m, 4H, H_(Ar), H-6), 8.42(t, J=13.4 Hz, ═CH). ³¹P NMR (D₂O) δ −4.85 (m, 1P, P_(γ), −9.86 (m, 1P,P_(α)), −20.40 (m, 1P, P_(β)). Mass (−ve electrospray) calcd forC₄₉H₆₄N₉O₂₂P₃S₄ 1351.23, found 1372 (M−2H+Na), 1270 [M−80], 1190[M−160].

5-[3-(2,2,2-Trifluoroacetamido)-prop-1-ynyl]-2′-deoxycytidine (7)

To a solution of 5-iodo-2′-deoxycytidine (10 g, 28.32 mmol) in DMF (200ml) in a light protected round bottom flask under Argon atmosphere, wasadded CuI (1.08 g, 5.67 mmol), triethylamine (7.80 ml, 55.60 mmol),2,2,2-trifluoro-N-prop-2-ynyl-acetamide (12.8 g, 84.76 mmol) and at lastPd(PPh)₃)₄ (3.27 g, 2.83 mmol). After 18 hours at room temperature,dowex bicarbonate (20 mg) was added and the mixture was stirred for afurther 1 h. Filtration and evaporation of the volatiles under reducedpressure gave a residue that was purified by flash chromatography onsilica gel (CH₂Cl₂, CH₂Cl₂:EtOAc 1:1, EtOAc:MeOH 9:1). The expectedproduct (7) was obtained as a beige solid in quantitative yield. ¹H NMR(D₂O) δ 2.24-2.17 (m, 1H, H-2′), 2.41-2.37 (m, 1H, H-2′), 3.68 (dd,J=12.5, 5.0 Hz, 1H, H-5′), 3.77 (dd, J=12.5, 3.2 Hz, 1H, H-5′), 3.99 (m,1H, H-4′), 4.27 (s, 2H, CH₂N), 4.34 (m, 1H, H-3′), 6.11 (t, J=6.3 Hz,1H, H-1′), 8.1 (br s, 1H, NH); MS (ES): m/z (%) (M−H) 375 (100).

5′-O-(tert-Butyldimethylsilyl)-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxycytidine(8)

To a solution of the starting material (7) (1.0 g, 2.66 mmol) andimidazole (200 mg, 2.93 mmol) in DMF (3.0 ml) at 0° C., was slowly addedTBDMSCl (442 mg, 2.93 mmol) in four portions over 1 h. After 2 h, thevolatiles were evaporated under reduced pressure and the residue wasadsorbed on silica gel and purified by flash chromatography (EtOAc,EtOAc:MeOH 9.5:0.5). The expected product (8) was isolated as acrystalline solid (826 mg, 64%). ¹H NMR (d₆ DMSO) δ 0.00 (s, 1H, CH₃);0.01 (s, 1H, CH₃), 0.79 (s, 9H, tBu), 1.87-1.80 (m, 1H, H-2′), 2.12(ddd, J=13.0, 5.8 and 3.0 Hz, 1H, H-2′), 3.65 (dd, J=11.5, 2.9 Hz, 1H,H-5′), 3.74 (dd, J=11.5, 2.5 Hz, 1H, H-5′), 3.81-3.80 (m, 1H, H-4′),4.10-4.09 (m, 1H, H-3′), 4.17 (d, 2H, J=5.1 Hz, NCH₂), 5.19 (d, 1H,J=4.0 Hz, 3′-OH), 6.04 (t, J=6.6 Hz, 1H, H-1′), 6.83 (br s, 1H, NHH),7.78 (br s, 1H, NRH), 7.90 (s, 1H, H-6), 9.86 (t, J=5.1 Hz, 1H, —H₂CNH);MS (ES): m/z (%) (MH)⁺ 491 (40%).

4-N-Acetyl-5′-O-(tert-butyldimethylsilyl)-3′-O-(methylthiolmethyl)-5-[3-(2,2,2-trifluoroacetamide)-prop-1-ynyl]-2′-deoxycytidine(9)

To a solution of the starting material (8) (825 mg, 1.68 mmol) in DMSO(6.3 ml) and N₂ atmosphere, was slowly added acetic acid (AcOH) (1.3 ml,23.60 mmol) followed by acetic anhydride (Ac₂O) (4.8 ml, 50.50 mmol).The solution was stirred at room temperature for 18 h and quenched at 0°C. by addition of saturated NaHCO₃ (20 ml). The product was extractedinto EtOAc (3×30 ml), organic extracts combined, dried (MgSO₄), filteredand the volatiles evaporated. The crude residue was purified by flashchromatography on silica gel (EtOAc:petroleum ether 1:1) to give theexpected product as a colourless oil (9) (573 mg, 62%). ¹H NMR (d₆ DMSO)δ 0.00 (s, 6H, 2×CH₃), 0.78 (s, 9H, tBu), 2.01 (s, 3H, SCH₃), 2.19-1.97(m, 2H, 2×H2′), 2.25 (s, 3H, COCH₃), 3.67 (dd, 1H, J=11.5 Hz, H-5′),3.78 (dd, 1H, J=11.5, 3.3 Hz, H-5′), 4.06-4.05 (m, 1H, H-4′), 4.17 (d,2H, J=5.1 Hz, N—CH₂), 4.30-4.28 (m, 1H, H-3′), 4.63 (s, 2H; CH₂—S), 5.94(t, 1H, J=6.5 Hz, H-1′), 8.17 (s, 1H, H-6), 9.32 (s, 1H, NHCO), 9.91 (t,1H, J=5.4 Hz, NHCH₂); MS (ES): m/z (%) (MH)⁺ 593.

4-N-Acetyl-3′-O-(azidomethyl)-5′-O-(tert-butyldimethylsilyl)-5-[3-(2,2,2-trifluoroacetamide)-prop-1-ynyl]-2′-deoxycytidine(10)

To a solution of the starting material (9) (470 mg, 0.85 mmol) indicloromethane (DCM) (8 ml) under N₂ atmosphere and cooled to 0° C., wasadded cyclohexene (430 μl, 4.27 mmol) followed by SO₂Cl₂ (1 M in DCM,1.0 ml, 1.02 mmol). The solution was stirred for 30 minutes at 0° C.,and the volatiles were evaporated. Residue immediately dissolved in DMF(8 ml) stirred under N₂ and sodium azide (275 mg, 4.27 mmol) slowlyadded. After 18 h, the crude product was evaporated to dryness,dissolved in EtOAc (30 ml) and washed with Na₂CO₃ (3×5 ml). The combinedorganic layer was kept separately. A second extraction of the productfrom the aqueous layer was performed with DCM (3×10 ml). All thecombined organic layers were dried (MgSO₄), filtered and the volatilesevaporated under reduced pressure to give an oil identified as theexpected product (10) (471 mg, 94% yield). This was used without anyfurther purification. ¹H NMR (d₆ DMSO) δ 0.11 (s, 3H, CH₃), 0.11 (s, 3H,CH₃), 0.88 (s, 9H, ^(t)Bu), 2.16-2.25 (m, 1H, H-2′), 2.35 (s, 3H,COCH₃), 2.47-2.58 (m, 1H, H-2′), 3.79 (dd, J=11.6, 3.2 Hz, 1H, H-5′),3.90 (dd, J=11.6, 3.0 Hz, 1H, H-5′), 4.17-4.19 (m, 1H, H-4′), 4.28 (s,2H, NCH₂), 4.32-4.35 (m, 1H, H-3′), 4.89 (dd, J=14.4, 6.0 Hz, 2H,CH₂—N₃), 6.05 (t, J=6.4 Hz, 1H, H-1′), 8.25 (s, 1H, H-6), 9.46 (br s,1H, NHH), 10.01 (br s, 1H, NHH).

4-N-Acetyl-3′-O-(azidomethyl)-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxycytidineand3′-O-(Azidomethyl)-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxycytidine(11)

To a solution of the starting material (11) (440 mg, 0.75 mmol) in THF(20 ml) at 0° C. and N₂ atmosphere, was added TBAF in THF 1.0 M (0.82ml, 0.82 mmol). After 1.5 h, the volatiles were evaporated under reducedpressure and the residue purified by flash chromatography on silica gel(EtOAc:petroleum ether 8:2 to EtOAc 100% to EtOAc:MeOH 8:2). Twocompounds were isolated and identified as above described. The firsteluted 4-N-Acetyl (11), (53 mg, 15%) and, the second one 4-NH₂ (12) (271mg, 84%).

Compound 4-N-Acetyl (11): ¹H NMR (d₆ DMSO) δ 1.98 (s, 3H, CH₃CO),2.14-2.20 (m, 2H, HH-2′), 3.48-3.55 (m, 1H, H-5′), 3.57-3.63 (m, 1H,H-5′), 3.96-4.00 (m, 1H, H-4′), 4.19 (d, J=5.3 Hz, 2H, CH₂—NH),4.23-4.28 (m, 1H, H-3′), 4.77 (s, 2H, CH₂—N₃), 5.2 (t, 1H, J=5.1 Hz,5′-OH), 5.95 (t, J=6.2 Hz, 1H, H-1′), 8.43 (s, 1H, H-6), 9.34 (s, 1H,CONH), 9.95 (t, J=5.3 Hz, 1H, NHCH₂).

Compound 4-NH₂ (12): ¹H NMR (d₆ DMSO) δ 1.98-2.07 (2H, CHH-2′),3.50-3.63 (m, 2H, CHH-5′), 3.96-4.00 (m, 1H, H-4′), 4.09 (d, J=5.3 Hz,2H, CH₂—NH), 4.24-4.28 (m, 1H, H-3′), 4.76 (s, 2H, CH₂—N₃), 5.13 (t,J=5.3 Hz, 1H, 5′-OH), 5.91 (br s, 1H, NHH), 6.11 (t, J=6.4 Hz, 1H,H-1′), 8.20 (t, J=5.3 Hz, 1H, NCH₂), 8.45 (s, 1H, H-6), 11.04 (br s, 1H,NHH).

4-N-Benzoyl-5′-O-(tert-butyldimethylsilyl)-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxycytidine(13)

The starting material (8) (10 g, 20.43 mmol) was azeotroped in drypyridine (2×100 ml) then dissolved in dry pyridine (160 ml) under N₂atmosphere. Chlorotrimethylsilane (10 ml, 79.07 mmol) added drop wise tothe solution and stirred for 2 hours at room temperature. Benzoylchloride (2.6 ml, 22.40 mmol) was then added to solution and stirred forone further hour. The reaction mixture was cooled to 0° C., distilledwater (50 ml) added slowly to the solution and stirred for 30 minutes.Pyridine and water were evaporated from mixture under high vacuum toyield a brown gel that was portioned between 100 ml of sat. aq. NaHCO₃(100 ml) solution DCM. The organic phase was separated and the aqueousphase extracted with a further (2×100 ml) of DCM. The organic layerswere combined, dried (MgSO₄), filtered and the volatiles evaporatedunder reduced pressure. The resulting brown oil was purified by flashchromatography on silica gel (DCM:MeOH 99:1 to 95:5) to yield a lightyellow crystalline solid (13) (8.92 g, 74%): ¹H NMR (d₆ DMSO): b 0.00(s, 6H, CH₃), 0.78 (s, 9H, tBu), 1.94 (m, 1H, H-2′), 2.27 (m, 1H, H-2′),3.64 (d, 1H, J=11.6 Hz, H-5′), 3.75 (d, 1H, J=11.6 Hz, H-5′), 3.91 (m,1H, H-4′), 4.09 (br m, 3H, CH₂NH, H-3′), 5.24 (s, 1H, 3′-OH), 6.00 (m,1H, H-1′), 7.39 (m, 2H, Ph), 7.52 (m, 2H, Ph), 7.86 (m, 1H, Ph), 8.0 (s,1H, H-6), 9.79 (t, 1H, J=5.4 Hz, NHCH₂), 12.67 (br s, 1H, NH). Mass (+veelectrospray) calcd for C₂₇H₃₃P₃N₄O₆Si 594.67, found 595.

4-N-Benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-O-methylthiomethyl-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxycytidine(14)

The starting material (13) (2.85 g, 4.79 mmol) was dissolved in dry DMSO(40 ml) under N₂ atmosphere. Acetic acid (2.7 ml, 47.9 mmol) and aceticanhydride (14.4 ml, 143.7 mmol) were added sequentially and slowly tothe starting material, which was then stirred for 18 h at roomtemperature. Saturated NaHCO₃ (150 ml) solution was carefully added tothe reaction mixture. The aqueous layer was extracted with EtOAc (3×150ml). The organic layers were combined, dried (MgSO₄), filtered andevaporated to yield an orange liquid that was subsequently azeotropedwith toluene (4×150 ml) until material solidified. Crude residuepurified on silica gel (petroleum ether:EtOAc 3:1 to 2:1) to yield ayellow crystalline solid (14) (1.58 g, 50%). ¹H NMR (d₆ DMSO): δ 0.00(s, 6H, CH₃), 0.78 (s, 9H, tBu), 1.99 (s, 3H, CH₃), 2.09 (m, 1H, H-2′),2.28 (m, 1H, H-2′), 3.66 (d, 1H, J=11.5, 2.9 Hz, H-5′), 3.74 (dd, 1H,J=11.3, 2.9 Hz, H-5′), 3.99 (m, 1H, H-4′), 4.09 (m, 1H, CH₂NH), 4.29 (m,1H, H-3′), 4.61 (s, 2H, CH₂S), 6.00 (m, 1H, H-1′), 7.37 (m, 2H, Ph),7.50 (m, 2H, Ph), 7.80 (d, 1H, J=7.55 Hz, H_(Ar)), 7.97 (s, 1H, H-6),9.79 (br t, 1H, NHCH₂), 12.64 (br s, 1H, NH). Mass (−ve electrospray)calcd for C₂₉H₃₇F₃N₄O₆SSi 654.79, found 653.2.

4-N-Benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-O-azidomethyl-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxycytidine(15)

The starting material (14) (1.65 g, 2.99 mmol) was dissolved in DCM (18ml) and cooled to 0° C. Cyclohexene (1.5 ml, 14.95 mmol) and SO₂Cl₂(0.72 ml, 8.97 mmol) were added and stirred 1 h in ice bath. TLCindicated starting material still to be present whereupon a furtheraliquot of SO₂Cl₂ (0.24 ml) was added and the mixture stirred for 1 h at0° C. Volatiles were removed by evaporation to yield a light brown solidthat was redissolved in 18 ml of dry DMF (18 ml) under N₂. Sodium azide(0.97 g, 14.95 mmol) was then added to the solution and stirred for 2.5h at room temperature. The reaction mixture was passed through a pad ofsilica and eluted with EtOAc and the volatiles removed by high vacuumevaporation. The resulting brown gel was purified by flashchromatography (petroleum ether:EtOAc 4:1 to 2:1) to yield the desiredproduct as a white crystalline solid (15) (0.9 g, 55%). ¹H NMR (d₆DMSO): δ 0.00 (s, 6H, CH₃), 0.78 (s, 9H, tBu), 2.16 (m, 1H, H-2′), 2.22(m, 1H, H-2′), 3.70 (d, 1H, J=11.5 Hz, H-5′), 3.75 (d, 1H, J=11.3 Hz,H-5′), 4.01 (m, 1H, H-4′), 4.10 (m, 1H, CH₂NH), 4.23 (m, 1H, H-3′), 4.76(s, 2H, CH₂S), 5.99 (m, 1H, H-1′), 7.37 (m, 2H, Ph), 7.50 (m, 2H, Ph),7.81 (d, 1H, J=7.4 Hz, Ph), 7.95 (s, 1H, H-6), 9.78 (br s, 1H, NHCH₂),12.64 (br s, 1H, NH). Mass (−ve electrospray) calcd. for C₂₈H₃₄F₃N₇O₆Si649.71, found 648.2

4-N-Benzoyl-3′-O-azidomethyl-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxycytidine(16)

The starting material (15) (140 mg, 0.22 mmol) was dissolved in THF (7.5ml). TBAF (1M soln. in THF, 0.25 ml) was added slowly and stirred for 2h at room temperature. Volatile material removed under reduced pressureto yield a brown gel that was purified by flash chromatography(EtOAc:DCM 7:3) to yield the desired product (16) as a light colouredcrystalline solid (0.9 g, 76%). ¹H NMR (d₆ DMSO): δ 2.16 (m, 1H, H-2′),2.22 (m, 1H, H-2′), 3.70 (d, 1H, J=11.5 Hz, H-5′), 3.75 (d, 1H, J=11.3Hz, H-5′), 4.01 (m, 1H, H-4′), 4.10 (m, 1H, CH₂NH), 4.23 (m, 1H, H-3′),4.76 (s, 2H, CH₂S), 5.32 (s, 1H, 5′ OH), 5.99 (m, 1H, H-1′), 7.37 (m,2H, Ph), 7.50 (m, 2H, Ph), 7.81 (d, 1H, J=7.35 Hz, Ph), 7.95 (s, 1H,H-6), 9.78 (br s, 1H, NHCH₂), 12.64 (br s, 1H, NH). Mass (−veelectrospray) calcd for C₂₂H₂₀F₃N₇O₆ 535.44, found 534.

5-(3-Amino-prop-1-ynyl)-3′-O-azidomethyl-2′-deoxycytidine5′-O-nucleoside triphosphate (17)

To a solution of (11) and (12) (290 mg, 0.67 mmol) and proton sponge(175 mg, 0.82 mmol) (both previously dried under P₂O₅ for at least 24 h)in PO(OMe)₃ (600 μl), at 0° C. under Argon atmosphere, was slowly addedPOCl₃ (freshly distilled) (82 μl, 0.88 mmol). The solution wasvigorously stirred for 3 h at 0° C. and then quenched by addition oftetra-tributylammonium diphosphate (0.5 M) in DMF (5.2 ml, 2.60 mmol),followed by nBu₃N (1.23 ml, 5.20 mmol) and triethylammonium bicarbonate(TEAB) 0.1 M (20 ml). After 1 h at room temperature aqueous ammoniasolution (ρ 0.88, 20 ml) was added to the mixture. Solution stirred atroom temperature for 15 h, volatiles evaporated under reduced pressureand the residue was purified by MPLC with a gradient of TEAB from 0.05Mto 0.7M. The expected triphosphate was eluted from the column at approx.0.60 M TEAB. A second purification was done by HPLC in a Zorbax SB-C18column (21.2 mm i.d.×25 cm) eluted with 0.1M TEAB (pump A) and 30% CH₃CNin 0.1M TEAB (pump B) using a gradient as follows: 0-5 min 5% B, Φ.2 ml;5-25 min 80% B, Φ.8 ml; 25-27 min 95% B, Φ.8 ml; 27-30 min 95% B, Φ.8ml; 30-32 min 5% B, Φ.8 ml; 32-35 min 95% B, Φ.2 ml, affording theproduct described above with a r_(t)(17): 20.8 (14.5 μmols, 2.5% yield);³¹P NMR (D₂O, 162 MHz) δ −5.59 (d, J=20.1 Hz, P_(χ)), −10.25 (d, J=19.3Hz, 1P, P_(α)), −20.96 (t, J=19.5 Hz, 1P, Pβ); ¹H NMR (D₂O) δ 2.47-2.54(m, 1H, H-2′), 2.20-2.27 (m, 1H, H-2′), 3.88 (s, 2H, CH₂N), 4.04-4.12(m, 1H, HH-5′), 4.16-4.22 (m, 1H, 4.24-4.30 (m, 1H, H-4′), 4.44-4.48 (m,1H, H-3′), 6.13 (t, J=6.3 Hz, 1H, H-1′), 8.35 (s, 1H, H-6); MS (ES): m/z(%) (M−H) 574 (73%), 494 (100%).

Alexa488 Disulfide Linker

Commercial available Alexa Fluor 488-NHS (35 mg, 54 μmol) was dissolvedin DMF (700 μL) and, to ensure full activation, 4-DMAP (7 mg, 59 μmol)and N,N′-disuccinimidyl carbonate (15 mg, 59 μmol) were sequentiallyadded. After 15 min on complete activation, a solution of the startingdisulfide (32.0 mg, 108 μmol) in DMF (300 μL) containingdiisopropylethylamine (4 μL) was added over the solution of theactivated dye. Further addition of diisopropylethylamine (20 μL) to thefinal mixture was done, ultrasonicated for 5 min and reacted for 18 h atroom temperature in the darkness. The volatiles were evaporated underreduced pressure and the crude residue was first purified passing itthrough a short ion exchange resin Sephadex-DEAE A-25 (40-120μ) column,first eluted with TEAB 0.1 M (25 ml) then 1.0 M TEAB (75 ml). The latestcontaining the two final compounds was concentrated and the residue wasHPLC purified in a Zorbax SB-C18 column (21.2 mm i.d.×25 cm) eluted with0.1M TEAB (pump A) and CH₃CN (pump B) using a gradient as follows: 0-2min 2% B, Φ.2 ml; 2-4 min 2% B, Φ.8 ml; 4-15 min 23% B, Φ.8 ml; 15-24min 23% B, Φ.8 ml; 24-26 min 95% B, Φ.8 ml; 26-28 min 95% B, Φ.8 ml,28-30 min 2% B, Φ.8 ml, 30-33 min 2% B, Φ.2 ml affording both compoundsdetailed above with t_(r): 19.0 (left regioisomer) and t_(r): 19.5(right regioisomer). Both regioisomers were respectively passed througha dowex ion exchange resin column, affording respectively 16.2 μmol and10.0 μmol, 62% total yield (based in commercial available Alexa Fluor488-NHS of 76% purity); ε₄₉₃=71,000 cm⁻¹ M⁻¹ in H₂O. ¹H NMR (D₂O) (leftregioisomer) δ 2.51 (t, J=6.8 Hz, 2H, CH₂), 2.66 (t, J=6.8 Hz, 2H, CH₂),2.71 (t, J=5.8 Hz, 2H, CH₂), 3.43 (t, J=5.8 Hz, 2H, CH₂), 6.64 (d, J=9.2HZ, 2H, H_(Ar)), 6.77 (d, J=9.2 Hz, 2H, H_(Ar)), 7.46 (s, 1H, H_(Ar)),7.90 (dd, J=8.1 and 1.5 Hz, 1H, H_(Ar)), 8.20 (d, J=8.1 Hz, 1H, H_(Ar)).¹H NMR (D₂O) (right regioisomer) δ 2.67 (t, J=6.8 Hz, 2H, CH₂), 2.82 (t,J=6.8 Hz, 2H, CH₂), 2.93 (t, J=6.1 Hz, 2H, CH₂), 3.68 (t, J=6.1 Hz, 2H,CH₂), 6.72 (d, J=9.3 HZ, 2H, H_(Ar)), 6.90 (d, J=9.3 HZ, 2H, H_(Ar)),7.32 (d, J=7.9 Hz, 1H, H_(Ar)), 8.03 (dd, J=7.9, 1.7 Hz, 1H, H_(Ar)),8.50 (d, J=1.8 Hz, 1H, H_(Ar)) Mass (−ve electrospray) calcd forC₂₆H₂₃N₃O₁₂S₄ 697.02, found 692 (M−H), 347 [M/2].

To a solution of Alexa Fluor 488 disulfide linker (3.4 μmol, 2.37 mg) inDMF (200 μL) was added 4-DMAP (0.75 mg, 5.1 μmol) and N,N-disuccinimidylcarbonate (1.70 mg, 5.1 μmol). The mixture was stirred for 15 to fullactivation of the acid, then it was added into the solution of thenucleotide (17) (3.45 mg, 6.0 μmol) in DMF (0.3 ml) containing nBu₃N (40μL) at 0° C. The mixture was sonicated for 3 min and then continuouslystirred for 16 h in the absence of light. The volatiles were evaporatedunder reduced pressure and the residue was firstly purified byfiltration through a short ion exchange resin Sephadex-DEAE A-25 column,first eluted with TEAB 0.1 M (50 ml) removing the unreacted dye-linker,then 1.0 M TEAB (100 ml) to collect the expected product (18). Afterconcentration and the residue was HPLC purified in a Zorbax SB-C18column (21.2 mm i.d.×25 cm) eluted with 0.1M TEAB (pump A) and CH₃CN(pump B) using a gradient as follows: 0-2 min 2% B, 0.2 ml; 2-4 min 2%B, Φ.8 ml; 4-15 min 23% B, Φ.8 ml; 15-24 min 23% B, Φ.8 ml; 24-26 min95% B, Φ.8 ml; 26-28 min 95% B, Φ.8 ml, 28-30 min 2% B, Φ.8 ml, 30-33min 2% B, Φ.2 ml affording the product detailed above with a r_(t)(18):19.8 (0.26 μmols, 12% yield based on UV measurement); λ_(max)=493 nm, ∈71,000 cm⁻¹ M⁻¹ in H₂O); ³¹P NMR (D₂O, 162 MHz) δ −5.06 (d, J=20.6 Hz,1P, P_(χ)), −10.25 (d, J=19.3 Hz, 1P, P_(α)), −21.21 (t, J=19.5 Hz, 1P,P_(β)); ¹H NMR (D₂O) δ 2.09-2.17 (m, 1H, HH-2′), 2.43-2.50 (m, 1H,HH-2′), 2.61 (t, J=6.8 Hz, 2H, H₂C—S), 2.83 (2H, S—CH₂), 3.68 (t, J=6.0Hz, 2H, ArCONCH₂), 4.06 (s, 2H, CH₂N), 4.08-4.17 (m, 4H, HH-5′),4.25-4.29 (m, 1H, H-4′), 4.46-4.50 (m, 1H, H-3′), 6.09 (t, J=6.4 Hz, 1H,H-1′), 6.88 (d, J=9.1 Hz, 1H, H_(Ar)), 6.89 (d, J=9.3 Hz, 1H, H_(Ar)),7.15 (d, J=9.3 Hz, 1H, H_(Ar)), 7.17 (d, J=9.1 Hz, 1H, H_(Ar)), 7.64 (brs, 1H, H_(Ar)), 8.00-7.94 (m, 2H, H_(Ar)) 8.04 (s, 1H, H-6); MS (ES):m/z (%) (M−H) 1253 (46%), (M−H+Na)⁻ 1275 (100%).

7-Deaza-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyguanosine(19)

Under N₂, a suspension of 7-deaza-7-iodo-guanosine (2 g, 2.75 mmol),Pd(PPh₃)₄ (582 mg, 0.55 mmol), CuI (210 mg, 1.1 mmol), Et₃N (1.52 ml, 11mmol) and the propagylamine (2.5 g, 16.5 mmol) in DMF (40 ml) wasstirred at room temperature for 15 h under N₂. The reaction wasprotected from light with aluminium foil. After TLC indicating the fullconsumption of starting material, the reaction mixture was concentrated.The residue was diluted with MeOH (20 ml) and treated with dowex-HCO₃ ⁻.The mixture was stirring for 30 min and filtered. The solution wasconcentrated and purified by silica gel chromatography (petroleumether:EtOAc 50:50 to petroleum ether:EtOAc:MeOH 40:40:20), giving (19)as a yellow powder (2.1 g, 92%). ¹H NMR (d₆ DMSO) δ 2.07-2.11 (m, 1H,H-2′), 2.31-2.33 (m, 1H, H-2′), 3.49-3.53 (m, 2H, H-5′), 3.77 (br s, 1H,H-4′), 4.25 (d, J=4.3 Hz, 2H, —CCH₂), 4.30 (br s, 1H, H-3′), 4.95 (t,J=5.2 Hz, 1H, 5′-OH), 5.25 (d, J=3.4 Hz, 1H, 3′-OH), 6.27-6.31 (m, 1H,H-1′), 6.37 (s, 2H, NH₂), 7.31 (s, 1H, H-8), 10.10 (br s, 1H, NHCOCF₃),10.55 (s, 1H, NH). Mass (−ve electrospray) calcd for C₁₆H₃₆F₃N₅O₅ 415,found 414.

5′-O-(tert-Butyldiphenyl)-7-deaza-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyguanosine(20)

A solution of (19) (2.4 g, 5.8 mmol) in pyridine (50 ml) was treatedwith tert-butyldiphenylsilyl chloride (TBDPSCl) (1.65 ml, 6.3 mmol) dropwise at 0° C. The reaction mixture was then warmed to room temperature.After 4 h, another portion of TBDPSCl (260 μL, 1 mmol) was added. Thereaction was monitored by TLC, until full consumption of the startingmaterial. The reaction was quenched with MeOH (˜5 ml) and evaporated todryness. The residue was dissolved in DCM and aq. sat. NaHCO₃ was added.The aqueous layer was extracted with DCM three times. The combinedorganic extracts were dried (MgSO₄) and concentrated under vacuum.Purification by chromatography on silica (EtOAc to EtOAc:MeOH 85:15)gave (20) a yellow foam (3.1 g, 82%). ¹H NMR (d₆ DMSO) δ 1.07 (s, 9H,CH₃), 2.19-2.23 (m, 1H, H-2′), 2.38-2.43 (m, 1H, H-2′), 3.73-3.93 (m,2H, H-5′), 4.29 (d, J=5.0 Hz, 2H, CH₂N), 4.42-4.43 (m, 1H, H-3′), 5.41(br s, 1H, OH), 6.37 (t, J=6.5 Hz, H-1′), 6.45 (br s, 2H, NH₂),7.24-7.71 (m, 11H, H-8, H_(Ar)) 10.12 (t, J=3.6 Hz, 1H, NH), 10.62 (s,1H, H-3). Mass (+ve electrospray) calcd for C₃₂H₃₄F₃N₅O₅Si 653, found654.

5′-O-(tert-Butyldiphenyl)-7-deaza-3′-O-methylthiolmethyl-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyguanosine(21)

A solution of (20) (1.97 g, 3.0 mmol) in DMSO (15 ml) was treated withAc₂O (8.5 ml, 90 mmol), and AcOH (2.4 ml, 42 mmol) and stirred at roomtemperature for 15 h, then 2 h at 40° C. The reaction mixture wasdiluted with EtOAc (200 ml) and stirred with sat, aq. NaHCO₃ (200 ml)for 1 h. The aqueous layer was washed with EtOAc twice. The organiclayer was combined, dried (MgSO₄) and concentrated under vacuum.Purification by chromatography on silica (EtOAc:Hexane 1:1 toEtOAc:Hexane:MeOH 10:10:1) gave (21) as a yellow foam (1.3 g, 60%). ¹HNMR (CDCl₃) δ 1.04 (s, 9H, CH₃), 2.08 (s, 3H, SCH₃), 2.19-2.35 (m, 2H,H-2), 3.67-3.71 (m, 2H, H-5′), 3.97-3.99 (m, 2H, H-4′, H-3′), 4.23 (brs, 2H, CH₂N), 4.58 (s, 2H; CH₂S), 6.31 (dd, J=5.7, 7.9 Hz, H-1′),7.19-7.62 (m, 11H, H8, H_(Ar)). Mass (+ve electrospray) calcd forC₃₄H₃₈F₃N₅O₅SSi 713, found: 714.

3′-O-Azidomethyl-7-deaza-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyguanosine(22)

To a solution of (21) (1.3 mg, 1.8 mmol), cyclohexene (0.91 ml, 9 mmol)in CH₂Cl₂ (10 ml) in 4° C., sulfurylchloride (1M in CH₂Cl₂) (1.1 ml, 1.1mmol) was added drop wise under N₂. After 30 min., TLC indicated thefull consumption of the nucleoside (22). After evaporation to remove thesolvent, the residue was then subjected to high vacuum for 20 min, andthen treated with NaN₃ (585 mmol, 9 mmol) and DMF (10 ml). The resultedsuspension was stirred under room temperature for 2 h. Extraction withCH₂Cl₂/NaCl (10%) gave a yellow gum, which was treated with TBAF in THF(1 M, 3 ml) and THF (3 ml) at room temperature for 20 min. Evaporationto remove solvents, extraction with EtOAc/sat. aq. NaHCO₃, followed bypurification by chromatography on silica (EtOAc to EtOAc:MeOH 9:1) gave(22) as a yellow foam (420 mg, 50%). ¹H NMR (d₆ DMSO): δ 2.36-2.42 (m,1H, H-2′), 2.49-2.55 (m, 1H, H-2′), 3.57-3.59 (m, 2H, H-5′), 3.97-4.00(m, 1H, H-4′), 4.29 (m, 2H, CH₂N), 4.46-4.48 (m, 1H, H-3′), 4.92-4.96(m, 2H, CH₂N₃), 5.14 (t, J=5.4 Hz, 1H, 5′-OH), 5.96-6.00 (dd, J=5.7, 8.7Hz, 1H, H-1′), 6.46 (br s, 2H, NH₂), 7.39 (s, 1H, H-6), 10.14 (s, 1H,NH), 10.63 (s, 1H, H-3).

3′-O-Azidomethyl-7-deaza-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyguanosine5′-O-nucleoside triphosphate (23)

Tetrasodium diphosphate decahydrate (1.5 g, 3.4 mmol) was dissolved inwater (34 ml) and the solution was applied to a column of dowex 50 inthe H⁺ form. The column was washed with water. The eluent droppeddirectly into a cooled (ice bath) and stirred solution of tri-n-butylamine (1.6 ml, 6.8 mmol) in EtOH (14 ml). The column was washed untilthe pH of the eluent increased to 6. The aqueous ethanol solution wasevaporated to dryness and then co-evaporated twice with ethanol andtwice with anhydrous DMF. The residue was dissolved in DMF (6.7 ml). Thepale yellow solution was stored over 4 Å molecular sieves. Thenucleoside (22) and proton sponge was dried over P₂O₅ under vacuumovernight. A solution of (22) (104 mg, 0.22 mmol) and proton sponge (71mg, 0.33 mmol) in trimethylphosphate (0.4 ml) was stirred with 4 Åmolecular sieves for 1 h. Freshly distilled POCl₃ (25 μl, 0.26 mmol) wasadded and the solution was stirred at 4° C. for 2 h. The mixture wasslowly warmed up to room temperature and bis (tri-n-butyl ammonium)pyrophosphate (1.76 ml, 0.88 mmol) and anhydrous tri-n-butyl amine (0.42ml, 1.76 mmol) were added. After 5 min, the reaction was quenched with0.1 M TEAB (triethylammonium bicarbonate) buffer (15 ml) and stirred for3 h. The water was removed under reduced pressure and the resultingresidue dissolved in concentrated ammonia (ρ 0.88, 10 ml) and stirred atroom temperature for 16 h. The reaction mixture was then evaporated todryness. The residue was dissolved in water and the solution applied toa DEAE-Sephadex A-25 column. MPLC was performed with a linear gradientof 2 L each of 0.05 M and 1 M TEAB. The triphosphate was eluted between0.7 M and 0.8 M buffer. Fractions containing the product were combinedand evaporated to dryness. The residue was dissolved in water andfurther purified by HPLC. t_(r)(23)=20.5 min (Zorbax C18 preparativecolumn, gradient: 5% to 35% B in 30 min, buffer A 0.1M TEAB, buffer BMeCN). The product was isolated as a white foam (225 O.D., 29.6 μmol,13.4%, ε₂₆₀=7,600). ¹HNMR (D₂O) δ 2.43-2.5 (m, 2H, H-2′), 3.85 (m, 2H,CH₂N), 3.97-4.07 (m, 2H, H-5′), 4.25 (br s, 1H, H-4′), 4.57 (br s, 1H,H-3′), 4.74-4.78 (m, 2H, CH₂N₃), 6.26-6.29 (m, 1H, H-1′), 7.41 (s, 1H,H-8). ³¹P-NMR (D₂O) δ −8.6 (m, 1P, P_(γ)), −10.1 (d, J=19.4 Hz, 1P,P_(α)), −21.8 (t, J=19.4 Hz, 1P, P_(β)). Mass (−ve electrospray) calcdfor C₁₅H₂₁N₈O₁₃P₃ 614, found 613.

A mixture of disulphide linkered-Cy3 (2.5 μmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.95mg, 5 μmol), 1-hydroxybenzotriazole (HOBt) (0.68 mg, 5 μmol) andN-methyl-morpholine (0.55 μL, 5 μmol) in DMF (0.9 ml) was stirred atroom temperature for 1 h. A solution of (23) (44 O.D., 3.75 μmol) in 0.1ml water was added to the reaction mixture at 4° C., and left at roomtemperature for 3 h. The reaction was quenched with TEAB buffer (0.1M,10 ml) and loaded on a DEAE Sephadex column (2×5 cm). The column wasfirst eluted with 0.1 M TEAB buffer (100 ml) and then 1 M TEAB buffer(100 ml). The desired triphosphate product was eluted out with 1 M TEABbuffer. Concentrating the fraction containing the product and applied toHPLC. t_(r)(24)=23.8 min (Zorbax C18 preparative column, gradient: 5% to55% B in 30 min, buffer A 0.1M TEAB, buffer B MeCN). The product wasisolated as a red foam (0.5 μmol, 20%, ε_(max)=150,000). ¹H NMR (D₂O) δ1.17-1.71 (m, 20H, 4×CH₂, 4×CH₃), 2.07-2.15 (m, 1H, H-2′), 2.21-2.30 (m,1H, H-2′), 2.52-2.58 (m, 2H, CH₂), 2.66-2.68 (m, 2H, CH₂), 2.72-2.76 (m,2H, CH₂), 3.08-3.19 (m, 2H, CH₂), 3.81-3.93 (m, 6H, CH₂, H-5′),4.08-4.16 (m, 1H, H-4′), 4.45-4.47 (m, 1H, H-3′), 4.70-4.79 (m, 2H,CH₂N₃), 6.05-6.08 (m, 2H, H_(Ar)), 6.15-6.18 (m, 1H, H-1′), 7.11 (s, 1H,H-8), 7.09-7.18 (m, 2H, CH), 7.63-7.72 (m, 4H, H_(Ar)), 8.27-8.29 (m,1H, CH). ³¹P NMR (D₂O) δ −4.7 (m, 1P, P_(γ)), −9.8 (m, 1P, P_(α)), −19.7(m, 1P, P_(β)). Mass (−ve electrospray) calcd forC₅₁H₆₆N₁₁O₂₁P₃S₄1389.25, found 1388 (M−H), 694 [M−2H], 462 [M−3H].

7-Deaza-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyadenosine(25)

To a suspension of 7-deaza-7-iodo-2′-deoxyadenosine (1 g, 2.65 mmol) andCuI (100 mg, 0.53 mmol) in dry DMF (20 ml) was added triethylamine (740μl, 5.3 mmol). After stirring for 5 mintrifluoro-N-prop-2-ynyl-acetamide (1.2 g, 7.95 mmol) and Pd(PPh₃)₄ (308mg, 0.26 mmol) were added to the mixture and the reaction was stirred atroom temperature in the dark for 16 h. MeOH (40 ml) and bicarbonatedowex was added to the reaction mixture and stirred for 45 min. Themixture was filtered. The filtrate washed with MeOH and the solvent wasremoved under vacuum. The crude mixture was purified by chromatographyon silica (EtOAc to EtOAc:MeOH 95:20) to give slightly yellow powder(25) (1.0 g, 95%). ¹H NMR (d₆ DMSO) δ 2.11-2.19 (m, 1H, H-2′), 2.40-2.46(m, 1H, H-2′), 3.44-3.58 (m, 2H, H-5′), 3.80 (m, 1H, H-4′), 4.29 (m, 3H,H-3′, CH₂N), 5.07 (t, J=5.5 Hz, 1H, OH), 5.26 (d, J=4.0 Hz, 1H, OH),6.45 (dd, J=6.1, 8.1 Hz, 1H, H-1′), 7.74 (s, 1H, H-8), 8.09 (s, 1H,H-2), 10.09 (t, J=5.3 Hz, 1H, NH).

5′-O-(tert-Butyldiphenylsilyl)-7-deaza-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyadenosine(26)

The nucleoside (25) (1.13 g, 2.82 mmol) was coevaporated twice in drypyridine (2×10 ml) and dissolved in dry pyridine (18 ml). To thissolution was added t-butyldiphenylsilylchloride (748 μl, 2.87 mmol) insmall portions at 0° C. The reaction mixture was let to warm up at roomtemperature and left stirring overnight. The reaction was quenched withsat. aq. NaCl solution. EtOAc (25 ml) was added to reaction mixture andthe aqueous layer was extracted with EtOAc three times. After drying thecombined organic extracts (MgSO₄) the solvent was removed under vacuum.Purification by chromatography on silica (DCM then EtOAc to EtOAc:MeOH85:15) gave (26) as a slightly yellow powder (1.76 g, 97%). ¹H NMR (d₆DMSO) δ 1.03 (s, 9H, tBu), 2.25-2.32 (m, 1H, H-2′), 2.06-2.47 (m, 1H,H-2′), 3.71-3.90 (m, 2H, H-5′), 3.90-3.96 (m, 1H, H-4′), 4.32 (m, 2H,CH₂N), 4.46 (m, 1H, H-3′), 5.42 (br s, 1H, OH), 6.53 (t, J=6.7 Hz, 1H,H-1′), 7.38-7.64 (m, 11H, H-8 and H_(Ar)), 8.16 (s, 1H, H-2), 10.12 (t,J=5.3 Hz, 1H, NH).

5′-O-(tert-Butyldiphenylsilyl)-7-deaza-4-N,N-dimethylformadin-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyadenosine(27)

A solution of the nucleoside (26) (831 mg, 1.30 mmol) was dissolved in amixture of MeOH:N,N-dimethylacetal (30 ml: 3 ml) and stirred at 40° C.The reaction monitored by TLC, was complete after 1 h. The solvent wasremoved under vacuum. Purification by chromatography on silica(EtOAc:MeOH 95:5) gave (27) as a slightly brown powder (777 mg, 86%). ¹HNMR (d₆ DMSO) δ 0.99 (s, 9H, tBu), 2.22-2.29 (m, 1H, H-2′), 2.50-2.59(m, 1H, H-2′), 3.13 (s. 3H, CH₃), 3.18 (s. 3H, CH₃), 3.68-3.87 (m, 2H,H-5′), 3.88-3.92 (m, 1H, H-4′), 4.25 (m, 2H, CH₂N), 4.43 (m, 1H, H-3′),6.56 (t, J=6.6 Hz, 1H, H-1′), 7.36-7.65 (m, 10H, H_(Ar)), 7.71 (s, 1H,H-8), 8.33 (s, 1H, CH), 8.8 (s, 1H, H-2), 10.12 (t, J=5.3 Hz, 1H, NH).

5′-O-(tert-Butyldiphenylsilyl)-7-deaza-4-N,N′-dimethylformadin-3′-O-methylthiomethoxy-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyadenosine(28)

To a solution of (27) (623 mg, 0.89 mmol) in dry DMSO (8 ml) was addedacetic acid (775 μl, 13.35 mmol) and acetic anhydride (2.54 ml, 26.7mmol). The mixture was stirred overnight at room temperature. Thereaction was then poured into EtOAc and sat. aq. NaHCO₃ (1:1) solutionand stirred vigorously. The organic layer was washed one more time withsat. aq. NaHCO₃ and dried over MgSO₄. After removing the solvent underreduced pressure, the product (28) was purified by chromatography onsilica (EtOAc:petroleum ether 1:2, then EtOAc) yielding (28) (350 mg,52%). ¹H NMR (d₆ DMSO): δ, 1.0 (s, 9H, tBu), 2.09 (s, 3H, SCH₃),2.41-2.48 (m, 1H, H-2′), 2.64-2.72 (m, 1H, H-2′), 3.12 (s, 3H, CH₃),3.17 (s, 3H, CH₃), 3.66-3.89 (m, 2H, H-5′), 4.04 (m, 1H, H-4′), 4.26 (m,J=5.6 Hz, 2H, CH₂), 4.67 (m, 1H, H-3′), 4.74 (br s, 2H, CH₂), 6.49 (t,J=6.1, 8.1 Hz, 1H, H-1′), 7.37-7.48 (m, 5H, H_(Ar)), 7.58-7.67 (m, 5H,H_(Ar)), 7.76 (s, 1H, H-8), 8.30 (s, 1H, CH), 8.79 (s, 1H, H-2), 10.05(t, J=5.6 Hz, 1H, NH).

3′-O-Azidomethyl-5′-O-(tert-butyldiphenylsilyl)-7-deaza-4-N,N-dimethylformadin-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyadenosine(29)

To a solution of (28) (200 mg, 0.26 mmol) and cyclohexene (0.135 ml, 1.3mmol) in dry CH₂Cl₂ (5 ml) at 0° C., sulfurylchoride (32 μl, 0.39 mmol)was added under N₂. After 10 min, TLC indicated the full consumption ofthe nucleoside (28). The solvent was evaporated and the residue wassubjected to high vacuum for 20 min. It was then redissolved in dry DMF(3 ml), cooled to 0° C. and treated with NaN₃ (86 mg, 1.3 mmol). Theresulting suspension was stirred under room temperature for 3 h. Thereaction was partitioned between EtOAc and water. The aqueous phaseswere extracted with EtOAc. The combined organic extracts were combinedand dried over MgSO₄. After removing the solvent under reduced pressure,the mixture was purified by chromatography on silica (EtOAc) yielding anoil (29) (155 mg, 80%). ¹H NMR (d₆ DMSO): δ 0.99 (s, 9H, tBu), 2.45-2.50(m, 1H, H-2′), 2.69-2.78 (m, 1H, H-2′), 3.12 (s, 3H, CH₂), 3.17 (s, 3H,CH₂), 3.67-3.88 (m, 2H, H-5′), 4.06 (m, 1H, H-4′), 4.25 (m, 2H, CH₂),4.61 (m, 1H, H-3′), 4.84-4.97 (m, 2H, CH₂), 6.58 (t, J=6.6 Hz, 1H,H-1′), 7.35-7.47 (m, 5H, H_(Ar)), 7.58-7.65 (m, 5H, H_(Ar)), 7.77 (s,1H, H-8), 8.30 (s, 1H, CH), 8.79 (s, 1H, H-2), 10.05 (br s, 1H, NH).

3′-O-Azidomethyl-7-deaza-4-N,N-dimethylformadin-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyadenosine(30)

A solution of (29) (155 mg, 0.207 mmol) in solution in tetrahydrofuran(THF) (3 ml) was treated with TBAF (1 M in THF, 228 μl) at 0° C. Theice-bath was then removed and the reaction mixture stirred at roomtemperature. After 2 h-TLC indicated the full consumption of thenucleoside. The solvent was removed. Purification by chromatography onsilica (EtOAc:MeOH 95:5) gave (30) (86 mg, 82%) as a pale brown oil. ¹HNMR (d₆ DMSO) δ 2.40-2.48 (dd, J=8.1, 13.6 Hz, 1H, H-2′), 2.59-2.68 (dd,J=8.3, 14 Hz, 1H, H-2′), 3.12 (s, 3H, CH₃), 3.17 (s, 3H, CH₃), 3.52-3.62(m, 2H, H-5′), 4.02 (m, 1H, H-4′), 4.28 (d, J=5.6 Hz, 2H, CH₂NH), 4.47(m, 1H, H-3′), 4.89 (s, 2H, CH₂N₃), 5.19 (t, J=5.6 Hz, 1H, OH), 6.49(dd, J=8.1, 8.7 Hz, 1H, H-1′), 7.88 (s, 1H, H-8), 8.34 (s, 1H, CH), 8.80(s, 1H, H-2), 10.08 (s, 1H, NH).

7-(3-Aminoprop-1-ynyl)-3′-O-azidomethyl-7-deaza-2′-deoxyadenosine5′-O-nucleoside triphosphate (31)

The nucleoside (30) and proton sponge was dried over P₂O₅ under vacuumovernight. A solution of (30) (150 mg, 0.294 mmol) and proton sponge(126 mg, 0.588 mmol) in trimethylphosphate (980 μl) was stirred with 4 Åmolecular sieves for 1 h. Freshly distilled POCl₃ (36 μl, 0.388 mmol)was added and the solution was stirred at 4° C. for 2 h. The mixture wasslowly warmed up to room temperature and bis (tri-n-butyl ammonium)pyrophosphate 0.5 M solution in DMF (2.35 ml, 1.17 mmol) and anhydroustri-n-butyl amine (560 μl, 2.35 mmol) was added. After 5 min, thereaction was quenched with 0.1 M TEAB (triethylammonium bicarbonate)buffer (15 ml) and stirred for 3 h. The water was removed under reducedpressure and the resulting residue dissolved in concentrated ammonia (p0.88, 15 ml) and stirred at room temperature for 16 h. The reactionmixture was then evaporated to dryness. The residue was dissolved inwater and the solution applied to a DEAE-Sephadex A-25 column. MPLC wasperformed with a linear gradient of 0.05 M to 1 M TEAB. Fractionscontaining the product were combined and evaporated to dryness. Theresidue was dissolved in water and further purified by HPLC. HPLC:t_(r)(31): 19.94 min (Zorbax C18 preparative column, gradient: 5% to 35%B in 20 min, buffer A 0.1M TEAB, buffer B MeCN). The product (31) wasisolated as a white foam (17.5 μmol, 5.9%, ε₂₈₀=15000). ¹H NMR (D₂O) δ2.67-2.84 (2m, 2H, H-2′), 4.14 (m, 2H, CH₂NH), 4.17-4.36 (m, 2H, H-5′),4.52 (br s, H-4′), 6.73 (t, J=6.6 Hz, H-1′), 8.06 (s, 1H, H-8), 8.19 (s,1H, H-2). ³¹P NMR (D₂O) δ −5.07 (d, J=21.8 Hz, 1P, P_(γ)), −10.19 (d,J=19.8 Hz, 1P, P_(α)), −21.32 (t, J=19.8 Hz, 1P, P_(β)). Mass (−veelectrospray) calcd for C₁₅H₂₁N₈O₁₂P₃ 598.05, found 596.

To the Cy3 disulphide linker (1.3 μmol) in solution in DMF (450 μl) isadded at 0° C. 50 μl of a mixture of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,1-hydroxybenzotriazole hydrate and N-methylmorpholine (26 μM each) inDMF. The reaction mixture was stirred at room temperature for 1 h. Thereaction was monitored by TLC (MeOH:CH₂Cl₂ 3:7) until all the dye linkerwas consumed. Then DMF (400 μl) was added at 0° C., followed by thenucleotide (31) (1.2 μmol) in solution in water (100 μl) and thereaction mixture and stirred at room temperature overnight. TLC(MeOH:CH₂Cl₂ 4:6) showed complete consumption of the activated ester anda dark red spot appeared on the baseline. The reaction was quenched withTEAB buffer (0.1M, 10 ml) and loaded on a DEAE Sephadex column (2×5 cm).The column was first eluted with 0.1 M TEAB buffer (100 ml) to wash offorganic residues and then 1 M TEAB buffer (100 ml). The desiredtriphosphate (32) was eluted out with 1 M TEAB buffer. The fractioncontaining the product were combined, evaporated and purified by HPLC.HPLC conditions: t_(r)(32): 22.44 min (Zorbax C18 preparative column,gradient: 5% to 35% B in 20 min, buffer A 0.1M TEAB, buffer B MeCN). Theproduct was isolated as dark pink solid (0.15 μmol, 12.5%, ε₅₅₀=150000).¹H NMR (D₂O) δ 2.03 (t, 2H, CH₂), 2.25 (m, 1H, H-2′), 2.43 (m, 1H,H-2′), 2.50 (m, 2H, CH₂), 2.66 (m, 2H, CH₂), 3.79 (m, 2H CH₂), 3.99 (m,4H, CH₂N, H-5′), 4.18 (br s, 1H, H-4′), 6.02, 6.17 (2d, J=13.64 Hz, 2H,H_(Ar)), 6.30 (dd, J=6.06, 8.58 Hz, H-1′), 7.08, 7.22 (2d, 2H, 2×═CH),7.58-7.82 (m, 5H, H_(Ar), H-2, H-8), 8.29 (m, ═CH). ³¹P NMR (D₂O) δ−4.83 (m, 1P, P_(γ)), −10.06 (m, 1P, P_(α)), −20.72 (m, 1P, P_(β)).

Enzyme Incorporation of 3′-Azidomethyl dNTPs

To a 100 nM DNA primer/template (primer previously labelled with P32 andT4 polynucleotide kinase) in Tris-HCl pH 8.8 50 mM, Tween-20 0.01%, andMgSO₄ 4 mM, add 2 μM compound 6 and 100 nM polymerase (Thermococcus sp.9° N exo ⁻Y409V A485L supplied by New England Biolabs). The templateconsists of a run of 10 adenine bases to show the effect of the block.The reaction is heated to 65 C for 10 mins. To show complete blocking, achase is performed with the four native, unblocked nucleosidetriphosphates. Quantitative incorporation of a single azidomethylblocked dTTP can be observed and thus the azidomethyl group can be seento act as an effective block to further incorporation.

By attaching a hairpin DNA (covalently attached self complementaryprimer/template) to a streptavidin bead The reaction can be performedover multiple cycles as shown in FIGS. 5 and 6.

Preparation of the Streptavidin Beads

Remove the storage buffer and wash the beads 3 times with TE buffer(Tris-HCl pH 8, 10 mM and EDTA, 1 mM). Resuspend in B & W buffer (10 mMTris-HCl pH 7.5, 1 mM EDTA and 2.0 M NaCl), add biotinylated ³²Plabelled hairpin DNA with appropriate overhanging template sequence.Allow to stand at room temperature for 15 minutes. Remove buffer andwash beads 3 times TE buffer.

Incorporation of the Fully Functional Nucleoside Triphosphate (FFN)

To a solution of Tris-HCl pH 8.8 50 mM, Tween-20 0.01%, MgSO₄ 4 mM,MnCl₂ 0.4 mM (except cycle 1, 0.2 mM), add 2 μM FFN and 100 nMpolymerase. This solution is then added to the beads and mixedthoroughly and incubated at 65° C. for 10-15 minutes. The reactionmixture is removed and the beads washed 3 times with TE buffer.

Deblocking Step

Tris-(2-carboxyethyl)phosphines trisodium salt (TCEP) (0.1M) is added tothe beads and mixed thoroughly. The mixture was then incubated at 65° C.for 15 minutes. The deblocking solution is removed and the beads washed3 times with TE buffer.

Capping Step

Iodoacetamide (431 mM) in 0.1 mM phosphate pH 6.5 is added to the beadsand mixed thoroughly, this is then left at room temperature for 5minutes. The capping solution is removed and the beads washed 3 timeswith TE buffer.

Repeat as required

The reaction products can be analysed by placing the bead solution inthe well of a standard 12% polyacrylamide DNA sequencing gel in 40%formamide loading buffer. Running the gel under denaturing conditionscauses the DNA to be released from the beads and onto the gel. The DNAband shifts are affected by both the presence of dye and the addition ofextra nucleotides and thus the cleavage of the dye (and block) with thephosphine cause a mobility shift on the gel.

Two cycles of incorporation with compounds 18 (C), 24 (G) and 32 (A) andsix cycles with compound 6 can be seen in figures FIG. 5 and FIG. 6.

3′-OH Protected with an Allyl Group

Nucleotides bearing this blocking group at the 3′position have beensynthesised, shown to be successfully incorporated by DNA polymerases,block efficiently and may be subsequently removed under neutral, aqueousconditions using water soluble phosphines or thiols allowing furtherextension.

5′-O-(t-Butyldimethylsilyl)-5-iodo-2′-deoxyuridine (33)

To a solution of 5-iodo-2′-deoxyuridine (5.0 g, 14 mmol) in 70 ml in dryN,N-dimethylformamide (DMF) was added imidazole (1.09 g, 16 mmol),followed by (2.41 g, 16 mmol) TBDMSCl at 0° C. The mixture was left inthe ice bath and stirred overnight. The reaction was quenched with sat.aq. NaCl solution and extracted with EtOAc. After drying (MgSO₄), thesolvent was removed and the crude mixture was purified by chromatographyon silica (EtOAc:petroleum ether 3:7). The product (33) (5.9 g, 90%) wasobtained as a colourless solid. ¹H NMR (d₆ DMSO) δ 0.00 (s, 3H, CH₃),0.79 (s, 9H, tBu), 1.88-1.97 (m, 1H, H-2′), 2.00-2.05 (m, 1H, H-2′),3.59-3.71 (m, 2H, H-5′), 3.75 (br s, 1H, H-4′), 4.06 (br s, 1H, H-3′),5.18 (d, J=4.0 Hz, 1H, OH), 5.98 (t, J=5.9 Hz, 1H, H-1′), 7.89 (s, 1H,H-6), 11.62 (s, 1H, NH). Mass (−ve electrospray) calcd for C₁₅H₂₅IN₂O₅Si468.06 found 467.

3′-O-Allyl-5′-O-t-butyldimethylsilyl-5-iodo-2′-deoxyuridine (34)

To a suspension of NaH (497 mg, 12.4 mmol, 60% in mineral oil) in dryTHF (20 ml) a solution of 5′-TBDMS protected 5-iodo-2′-deoxyuridine (2.8g, 5.9 mmol) in dry THF (50 ml) was added drop wise. After the gasevolution had stopped the mixture was stirred for another 10 min andthen allylbromide (561 μl, 6.5 mmol) was added drop wise. After thecomplete addition the milky reaction mixture was stirred at roomtemperature for 16 h. The reaction was quenched by addition of sat. aq.NaCl solution (30 ml). The aqueous layer was extracted three times usingEtOAc and after washing with sat. aq. NaCl solution the organic phasewas dried (MgSO₄). After removing of the solvents the crude product waspurified by chromatography (EtOAc:petroleum ether 1:1). The allylatedproduct (2.39 g, 80%) was obtained as a colourless foam. ¹H NMR (d₆DMSO) δ −0.01 (s, 3H, CH₃), 0.78 (s, 9H, tBu), 1.94-2.01 (m, 1H, H-2′),2.16-2.21 (m, 1H, H-2′), 3.61-3.71 (m, 2H, H-5′), 3.87-3.94 (m, 4H,H-3′, H-4′, OCH₂), 5.04 (dd, J=1.6, 10.4 Hz, 1H, ═CH₂), 5.15 (dd, J=1.8,17.3 Hz, 1H, ═CH₂), 5.72-5.81 (m, 1H, CH═), 5.92 (t, J=5.7 Hz, 1H,H-1′), 7.88 (s, 1H, 6-H), 11.6 (s, 1H, NH). Mass (−ve electrospray)calcd for C₁₈H₂₉IN₂O₅Si 508.09, found 507.

3′-O-Allyl-5-iodo-2′-deoxyuridine (35)

To a solution of (34) (2.34 g, 4.71 mmol) in dry THF (40 ml) was addedat 0° C. TBAF (5.2 ml, 5.2 mmol, 1 M solution in THF). The reactionmixture was allowed to warm up to room temperature and was then stirredfor 16 h. The reaction was quenched by adding sat. NaCl solution (20 ml)and extracted with EtOAc three times. The combined organic layers weredried over MgSO₄. The crude mixture was purified by chromatography onsilica (EtOAc:petrol 7:3). Product (35) (1.4 g, 75%) was isolated as acolourless solid. ¹H NMR (d₆ DMSO) δ 2.02-2.39 (m, 2H, H-2′), 3.42-3.52(m, 2H, H-5′), 3.84-3.88 (m, 3H, H-4′, CH₂], 3.97-4.00 (m, 1H, H-3′),5.02-5.09 (m, 2H, OH, ═CH₂), (dd, J=1.9, 17.3 Hz, 1H, ═CH₂), 5.73-5.82(m, 1H, CH═), 5.94 (t, J=6.8 Hz, 1H, H-1′), 8.24 (s, 1H, H-6), 11.56 (s,1H, NH). Mass (−ve electrospray) calcd for C₁₂H₁₆IN₂O₅ 394.0 found 393.

3′-O-Allyl-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyuridine

To a solution of (35) (400 mg, 1.0 mmol) in dry DMF (10 ml) was addedCuI (38 mg, 20 μmol) and triethylamine (300 μl, 2.0 mmol). Thepropargyltrifluoroacetamide (453 mg, 3.0 mmol) was added drop wise,followed by Pd(PPh₃)₄ (110 mg, 9.5 μmol). The reaction was stirred for16 h in the dark. The reaction was quenched by adding MeOH (10 ml), DCM(10 ml) and bicarbonate dowex. The mixture was stirred for 30 min andthen filtered. The solvents were removed under vacuum and the crudeproduct was purified by chromatography on silica (EtOAc:petrol 3:7 to7:3). The product was isolated as slightly yellow crystals (398 mg,95%). ¹H NMR (d₆ DMSO) δ 2.25-2.43 (m, 2H, H-2′), 3.65-3.76 (m, 2H,H-5′), 4.07-4.17 (m, 3H, H-4′, CH₂), 4.21-4.23 (m, 1H, H-3′), 4.34 (d,J=5.5 Hz, 2H, CH₂N), 5.25-5.27 (m, 2H, ═CH₂, OH), 5.38 (dd, J=1.83, 17.3Hz, 1H, ═CH₂), 5.96-6.06 (m, 1H, ═CH), 6.17 (t, J=6.9 Hz, 1H, H-1′),8.29 (s, 1H, H-6), 10.17 (t, J=5.5 Hz, 1H, NHTFA), 11.78 (s, 1H, NH).Mass (−ve electrospray) calcd for C₁₇H₁₈F₃N₃O₆ 417.11, found 416.

3′-O-Allyl-5-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyuridine5′-O-nucleoside triphosphate (37)

Under nitrogen (36) (100 mg, 0.24 mmol) and proton sponge (61.5 mg, 0.28mmol), both dried under vacuum over P₂O₅ for 24 h, were dissolved inOP(OMe)₃ (225 μl). At 0° C. freshly distilled POCl₃ was added drop wiseand the mixture was stirred for 1.5 h. Then pyrophosphate (1.44 ml, 0.72μmol, 0.5 M in DMF) and nBu₃N (0.36 ml, 1.5 mmol) were added and theresulting mixture stirred for another 1.5 h. Triethylammoniumbicarbonate solution (4.5 ml, 0.1 M solution, TEAB) was added and thereaction mixture was left stirring for 2 h. Then aq. NH₃ (4.5 ml) wasadded and the mixture was stirred for 16 h. After removing the solventsto dryness, the residue was redissolved in water, filtered and purifiedby MPLC, followed by HPLC purification. The desired triphosphate (37)(10.2 μmol, 4%, ε₂₈₀=10000) was isolated as a colourless foam. MPLCconditions: a gradient was run from 0.05M TEAB to 0.7 M TEAB using 2 lof each on a DEAE sephadex column. The product containing fractions cameoff with ˜0.4 M TEAB. After removing the solvent, the product was HPLCpurified. HPLC conditions: t_(r)(triphosphate): 21.9 min (Zorbax C-18preparative column, buffer A 0.1 M TEAB, buffer B 0.1 M TEAB+30%Acetonitrile, gradient 5-35% buffer B in 35 min). ¹H NMR (D₂O) δ2.17-2.23 (m, 1H, H-2′), 2.40-2.45 (m, 1H, H-2′), 3.67 (s, 2H, CH₂N),3.99 (d, J=5.9 Hz, 2H, OCH₂), 4.02-4.17 (m, 2H, H-5′), 4.25 (br s, 1H,H-4′), 4.32-4.33 (m, 1H, H-3′), 5.13 (d, J=10.3 Hz, 1H, ═CH₂), 5.23 (d,J=17.2 Hz, 1H, ═CH₂), 5.78-5.88 (m. 1H, ═CH), 6.16 (t, J=6.7 Hz, 1H,H-1′), 8.33 (s, 1H, H-6). ³¹P NMR (161.9 MHz, D₂O) δ −21.3 (t, J=19.5Hz, 1P, P_(γ)), −10.3 (d, J=19 Hz, 1P, P_(α)), −7.1 (d, J=15.5 Hz, 1P,P_(β)). Mass (−ve electrospray) calcd for C₁₅H₂₂N₃O₁₄P₃ 561.03, found560, 480 [M−phosphate], 401 [M−2× phosphate].

To a solution of Cy3 disulfide linker (2.5 μmol) in DMF (0.2 ml) at 0°C. was added. Disuccinimidyl carbonate (0.96 mg 3.75 μmol) and4-(dimethylamino) pyridine (DMAP) (0.46 mg 3.75 μmol). The reactionmixture was stirred for 10 min and then checked by TLC (MeOH:DCM 3:7)(activated ester r_(f)=0.5). In a separate flask the 3′-O-allylthymidine triphosphate (37) (532 μl, 14.1 mM in water, 7.5 μmol) weremixed with Bu₃N (143 μl) and evaporated to dryness. After this thetriphosphate (37) was dissolved in dry DMF (0.2 ml). To the triphosphate(37) solution at 0° C. was added the activated dye and the reactionmixture was allowed to warm to room temperature and then stirred for 16h. The solvent was removed and the residue was dissolved in water. Thereaction mixture was passed through a small DEAE sephadex column (2×5cm) using 0.1 M TEAB (100 ml) to remove the coupling reagents andunreacted linker. With 1 M TEAB (100 ml) the triphosphate (38) waseluted. The mixture was then separated by HPLC. Yield: 1.41 μmol (56%,ε₅₅₀=150000) product as a dark red solid were isolated. HPLC conditions:t_(r) (38): 19.6 min (Zorbax C-18 preparative column, buffer A 0.1 MTEAB, buffer B Acetonitrile, gradient: 2-58% buffer B in 29 min). ¹H (d₆DMSO) δ 0.75-0.79 (m, 3H, CH₃), 1.17-1.28 (m, 2H, CH₂), 1.48-1.55 (m,2H, CH₂), 1.64 (s, 12H, 4×CH₃), 1.70-1.77 (m, 2H, CH₂), 1.96-2.02 (m,1H, H-2′), 2.07-2.11 (m, 2H, CH₂), 2.25-2.30 (m, 1H, H-2′), 2.51-2.55(m, 2H, CH₂), 2.64-2.68 (m, 2H, CH₂), 2.75-2.81 (m, 2H, CH₂), 3.27-3.31(m, 2H, CH₂), 3.91-4.05 (m, 9H, H-5′, OCH₂, NCH₂, 2×NCH₂-dye), 4.13 (s,1H, H-4′), 4.22-4.24 (m, 1H, H-3′), 5.06 (d, J=10.5 Hz, 1H, ═CH₂), 5.15(dd, J=1.4 Hz, 17.3 Hz, 1H, ═CH₂), 5.72-5.82 (m, 1H, ═CH), 6.03-6.06 (m,1H, H-1′), 6.20-6.29 (m, 2H, αH), 7.23-7.31 (m, 2H, H_(Ar)), 7.63-7.79(m, 5H, H-6, 4×H_(Ar)), 8.31-8.45 (m, 1H, βH). ³¹P (161.9 MHz, d₆ DMSO)δ −20.2 (m, 1P, P_(β)), −10.0 (d, J 18.5 Hz, 1P, P_(α)), −4.8 (d, J 19.5Hz, 1P, P_(γ)). Mass (−ve electrospray) calcd for C₅₁H₆₇S₄N₆O₂₂P₃1336.24, found 1335.1, 688.1 [cleaved disulfide (dye), 647.9 [cleaveddisulfide (nucleotide)].

Enzyme Incorporation of Compound 38

To a 100 nM DNA primer/template (primer previously labelled with P32 andT4 polynucleotide kinase) in Tris-HCl pH 8.8 50 mM, Tween-20 0.01%, andMgSO₄ 4 mM, add 2 μM compound 38 and 100 nM polymerase (Thermococcus sp.9° N exo ⁻Y409V A485L supplied by New England Biolabs). The templateconsists of a run of 10 adenine bases to show the effect of the block.The reaction is heated to 65 C for 10 mins. To show complete blocking, achase is performed with the four native, unblocked nucleosidetriphosphates. Quantitative incorporation of the allyl block can beobserved (see FIG. 7) and this can be seen to act as an effective blockto further incorporation.

5′-O-(tert-Butyldimethylsilyl)-5-iodo-2′-deoxycytidine (39)

To a solution of 5-iodo-2′-deoxycytidine (2.2 g, 6.23 mmol) in DMF (130ml) was added imidazole (467 mg, 6.85 mmol). The mixture was cooled at0° C. and tert-butyldimethylsilyl chloride (TBDMSCl) (1.33 g, 6.85 mmol)added over 5 minutes. After 18 h at room temperature, the volatiles wereevaporated under reduced pressure and the residue purified by flashchromatography on silica gel with EtOAc:MeOH (95:5 to 90:10) to give theexpected product (39) (2.10 g, 72%) together with unreacted startingmaterial (490 mg). ¹H NMR (d₆ DMSO) δ 0.11 (s, 3H, CH₃), 0.12 (s, 3H,CH₃), 0.89 (s, 9H, 3CH₃), 1.90 (ddd, J=13.2, 7.7 and 5.7 Hz, 1H, 2.18(ddd, J=13.2, 5.7 and 2.3 Hz, 1H, HH-2′), 3.72 (dd, J=11.5, 3.6 Hz, 1H,HH-5′), 3.80 (dd, J=11.5, 2.8 Hz, 1H, HH-5′), 3.86-3.89 (m, 1H, H-4′),4.14-4.18 (m, 1H, H-3′), 5.22 (1H, d, J=4.1 Hz, OH), 6.09 (1H, dd,J=7.8, 5.8 Hz, H-1′), 6.60 (br s, 1H, NHH), 7.81 (br s, 1H, NRH), 7.94(s, 1H, H-6); MS (ES): m/z (%) (M+H) 468 (90%).

3′-O-Allyl-5′-O-(tert-butyldimethylsilyl)-5-iodo-2′-deoxycytidine (40)

To a solution of NaH (60%, 113 mg, 2.84 mmol) in THF (26 ml) under N₂atmosphere, was slowly added a solution of the starting nucleoside (39)(669 mg, 1.43 mmol) in THF (6 ml). The mixture was stirred at roomtemperature for 45 minutes, cooled at 0° C. and allyl bromide (134 μL,1.58 mmol) was slowly added. After 15 h at room temperature, thesolution was cooled to 0° C. and quenched by addition of H₂O (5 ml). THFevaporated under reduced pressure and the product extracted into EtOAc(3×25 ml). Combined organic extracts were dried (MgSO₄) filtered and thevolatiles evaporated under reduced pressure to give a residue that waspurified by flash chromatography on silica gel with EtOAc affording theexpected 3′-O-allyl product (40) (323 mg, 44%) as a colourless oil,together with some unreacted starting material (170 mg); ¹H NMR (d₆DMSO) δ 0.00 (s, 3H, CH₂), 0.01 (s, 3H, CH₂), 0.79 (s, 9H, 3CH₂), 1.84(ddd, J=13.3, 8.2 and 5.5 Hz, 1H, H-2′), 2.20-2.25 (m, 1H, H-2′),3.62-3.72 (m, 2H, H-5′), 3.88-3.93 (m, 4H, H-3′, 4′, HHC—CH═), 5.1 (dd,J=8.5, 1.7 Hz, 1H, CH═CHH), 5.16 (dd, J=17.2, 1.7 Hz, 1H, CH═CHH),5.75-5.83 (m, 1H, CH═CHH), 5.94 (dd, J=8.4, 5.6 Hz, 1H, H-1′), 6.53 (brs, 1H, NHH), 7.74 (br s, 1H, NHH), 7.83 (s, 1H, H-6); MS (ES): m/z (%)(M−H) 506 (100%).

3′-O-Allyl-5-iodo-2′-deoxycytidine (41)

To a solution of the starting nucleoside (40) (323 mg, 0.64 mmol) in THF(15 ml) under N₂ protected atmosphere was added at room temperaturetetrabutylammonium fluoride (TBAF) 1M in THF (0.7 ml, 0.7 mmol). Mixturestirred for one hour and then quenched by addition of H₂O (5 ml). THFwas evaporated and aqueous residue extracted into EtOAc (3×25 ml).Combined organic extracts were dried (MgSO₄), filtered and the volatilesevaporated under reduced pressure giving a crude material which waspurified by flash chromatography on a pre-packed silica column elutedwith EtOAc. The product (41) was obtained as a white solid (233 mg,93%). ¹H NMR (d₆ DMSO) δ 1.96-2.05 (m, 1H, H-2′) 2.24 (ddd, J=13.5, 5.8and 2.8 Hz, 1H, H-2′), 3.50-3.62 (m, 2H, H5′), 3.91-3.97 (m, 2H, H3′,H4′), 4.03-4.07 (m, 2H, HHC—CH═), 5.11-5.16 (m, 2H, OH, CH═CHH), 5.24(dd, J=17.2, 1.6 Hz, 1H, CH═CHH), 5.82-5.91 (m, 1H, CH═CHH), 6.02 (dd,J=7.6, 6.0 Hz, 1H, H-1′), 6.60 (s, 1H, NHH), 7.79 (s, 1H, NHH), 8.21 (s,1H, H-6). MS (ES): m/z (%) (M−H) 392 (100%).

3′-O-Allyl-5-[3-(2,2,2-trifluoroacetamide)-prop-1-ynyl]-2′-deoxycytidine(42)

To a solution of the starting nucleoside (41) (200 mg, 0.51 mmol) in dryDMF (8.5 ml) at room temperature and Argon atmosphere, was slowly addedCuI (19 mg, 0.10 mmol), NEt₃ (148 μl, 1.02 mmol),2,2,2-trifluoro-N-prop-2-ynyl-acetamide (230 mg, 1.53 mmol) andPd(PPh₃)₄ (58 mg, 0.05 mmol). The mixture was stirred at roomtemperature and protected from light during four hours, quenched byaddition of dowex bicarbonate and stirred for a 1 h, then filtered andthe volatiles evaporated under reduced pressure. The residue was furtherevaporated from MeOH (15 ml) and then purified by flash chromatographyon silica gel (CH₂Cl₂, CH₂Cl₂:EtOAc 1:1, EtOAc:MeOH 97.5:2.5). Theexpected product (42) was obtained as a beige solid (180 mg, 85%). ¹HNMR (d₆ DMSO) δ 1.90 (ddd, J=13.6, 7.7 and 6.0 Hz, 1H, H-2′), 2.16 (ddd,J=13.6, 5.7 and 2.4 Hz, 1H, H-2′), 3.42-3.50 (m, 2H, H-5′), 3.84-3.87(m, 3H, H-4′, OHHC—CH═), 3.94-3.96 (m, 1H, H-3′), 4.16 (d, J=5.1 Hz, 2H,H₂C—N), 4.98-5.05 (m, 2H, OH, CH═CHH), 5.14 (dd, J=17.3, 1.7 Hz, 1H,CH═CHH), 5.72-5.82 (m, 1H, CH═CHH), 5.95 (dd, J=7.7, 5.8 Hz, 1H, H-1′),6.74 (br s, 1H, NHH), 7.72 (br s, 1H, NHH), 8.01 (1H, s, H-6), 9.82 (brt, 1H, HN—CH₂). MS (ES): m/z (%) (M−H) 415 (100%).

3′-O-Allyl-5-(3-amino-prop-1-ynyl)-5′-O-triphosphate-2′-deoxycytidine(43)

To a solution of the nucleoside (42) (170 mg, 0.41 mmol) and protonsponge (105 mg, 0.50 mmol) (both previously dried under P₂O₅ for atleast 24 h) in PO(OMe)₃ (360 μl), at 0° C. under Argon atmosphere, wasslowly added POCl₃ (freshly distilled) (50 μl, 0.54 mmol). The solutionwas vigorously stirred for 3 h at 0° C. and then quenched by addition oftetra-tributylammonium diphosphate 0.5 M in DMF (3.20 ml, 1.60 mmol),followed by nBu₃N (0.75 ml, 3.2 mmol) and triethylammonium bicarbonate(TEAB) 0.1 M (12 ml). The mixture was stirred at room temperature for 3h and then an aqueous ammonia solution (ρ 0.88 1.0 ml) (12 ml) wasadded. The solution was stirred at room temperature for 15 h, volatilesevaporated under reduced pressure and the residue was purified by MPLCwith a gradient of TEAB from 0.05M to 0.7M. The expected triphosphate(43) was eluted from the column at approx. 0.51 M TEAB. A secondpurification was done by HPLC in a Zorbax SB-C18 column (21.2 mm i.d.×25cm) eluted with 0.1M TEAB (pump A) and 30% CH₃CN in 0.1M TEAB (pump B)using a gradient as follows: 0-5 min 5% B, Φ.2 ml; 5-25 min 80% B, Φ.8ml; 25-27 min 95% B, Φ.8 ml; 27-30 min 95% B, Φ.8 ml; 30-32 min 5% B,Φ.8 ml; 32-35 min 95% B, Φ.2 ml, affording the product (43) detailedabove with a t_(r)(43): 20.5 (20 μmols, 5% yield); ³¹P NMR (D₂O) δ −6.01(d, J=19.9 Hz, 1P, P_(γ)), −10.24 (d, J=19.3 Hz, 1P, P_(α)), −21.00 (t,J=19.6 Hz, 1P, P_(β)); ¹H NMR (D₂O) δ 2.19-2.26 (m, 1H, H-2′), 2.51 (1H,ddd, J=14.2, 6.1 and 3.2 Hz, H-2′)′, 3.96-4.07 (m, 4H, NCH₂, OHHC—CH═),4.09-4.14 (m, 1H, 1H, H-5′) 4.22-4.26 (m, 1H, H-5′), 4.30-4.37 (m, 2H,H-3′, 4′), 5.20 (d, J=10.4 Hz, 1H, CH═CHH), 5.30 (1H, dd, J=17.3, 1.5Hz, CH═CHH), 5.85-5.95 (m, 1H, CH═CHH), 6.18 (t, J=6.5 Hz, 1H, H-1′),8.40 (s, 1H, H-6); MS (ES): m/z (%) (M−H) 559 (100%).

To a solution of Alexa Fluor 488 disulfide linker (2.37 mg, 3.4 μmol) inDMF (500 μl) was added N,N-disuccinimidyl carbonate (1.3 mg, 5.1 μmol)and 4-DMAP (0.6 mg, 5.1 μmol). The mixture was stirred for 10 minutes,then it was added into the solution of the nucleotide (43) (3.23 mg, 5.8μmol) in DMF (100 μl) containing nBu₃N (30 μl). The mixture wascontinuously stirred for 16 h at room temperature. The volatiles wereevaporated under reduced pressure and the residue was firstly purifiedby passing it through a short ion exchange resin Sephadex-DEAE A-25(40-120μ)—column, first eluted with TEAB 0.1 M (70 ml) then 1.0 M TEAB(100 ml). The latest containing the expected product (44) wasconcentrated and the residue was HPLC purified in a Zorbax SB-C18 column(21.2 mm i.d.×25 cm) eluted with 0.1M TEAB (pump A) and CH₃CN (pump B)using a gradient as follows: 0-2 min 2% B, Φ.2 ml; 2-4 min 2% B, Φ.8 ml;4-15 min 23% B, Φ.8 ml; 15-24 min 23% B, Φ.8 ml; 24-26 min 95% B, Φ.8ml; 26-28 min 95% B, Φ.8 ml, 28-30 min 2% B, Φ.8 ml, 30-33 min 2% B, Φ.2ml affording the product detailed above with a r_(t)(44): 19.9 (0.56μmols, 17% yield based on UV measurement); λ_(max)=493 nm, ∈ 71,000 cm⁻¹M⁻¹ in H₂O); ³¹P NMR (D₂O) δ −5.07 (d, J=22.2 Hz, 1P, P_(χ)), −10.26 (d,J=19.4 Hz, 1P, P_(α)), −21.09 (t, J=19.7 Hz, 1P, P_(β)); ¹HNMR (D₂O) δ2.44-2.26 (m, 2H, HH-2′), 2.50 (t, J=6.7 Hz, 2H, CH₂), 2.83 (4H, CH₂,CH₂), 3.58 (t, J=6.0 Hz, 2H, CH₂), 4.07-3.91 (m, 6H, HH-5′, NCH₂,OHHC—CH═), 4.16-4.12 (m, 1H, H-4′), 4.23-4.17 (m, 1H, H-3′), 5.24-5.09(m, 2H, CH═CHH, CH═CHH), 5.84-5.74 (m, 1H, CH═CHH), 5.98 (t, J=8.1 Hz,1H, H-1′), 6.79 (d, J=9.1 Hz, 1H, H_(Ar)), 6.80 (d, J=9.3 Hz, 1H,H_(Ar)), 7.06 (t, J=8.8 Hz, 2H, H_(Ar)), 7.55 (br S, 1H, H_(Ar)),7.90-7.85 (m, 2H, H_(Ar)), 7.94 (s, 1H, H-6); MS (ES): m/z (%) (M−H)⁻1239 (27%).

5′-O-(tert-Butyldimethylsilyl)-7-deaza-7-iodo-2′-deoxyguanosine (45)

A solution of (44) (0.55 g, 1.4 mmol) in DMF (10 ml) was treated withimidazole (190 mg, 2.8 mmol) and TBDMSCl (274 mg, 1.82 mmol) at r.t. for15 h. The reaction was quenched with MeOH (˜5 ml). The mixture wasevaporated to dryness. Water (˜300 ml) was added to the residue andstirred for at least 1 h to fully dissolve imidazole. Filtration gave abrown solid, which was dried and purified by silica gel chromatography(DCM to DCM: MeOH 90:10), giving (45) as pale yellow powder (394 mg,56%). ¹H NMR (d₆ DMSO) δ 0.00, 0.01 (2s, 6H, CH₃), 0.82 (s, 9H, CH₃),1.99-2.05, 2.16-2.22 (2m, 2H, H-2′), 3.58-3.66 (m, 2H, H-5′), 3.72-3.74(m, 1H, H-4′), 4.18-4.19 (m, 1H, H-3′), 5.16 (d, J=3.0 Hz, 1H, OH), 6.20(dd, J=6.0, 8.0 Hz, 1H, H-1′), 6.25 (br s, 2H, NH₂), 7.58 (s, 1H, H-8),10.37 (s, 1H, HN). Mass (−ve electrospray) calcd for C₁₇H₂₇IN₄O₄Si 506,found 505.

3′-O-Allyl-5′-O-(tert-butyldimethylsilyl)-7-deaza-7-iodo-2′-deoxyguanosine(46)

A solution of (45) (354 mg, 0.7 mmol) in THF (25 ml) was treated withNaH (42 mg, 1.75 mmol) at r.t. for 1 h. Allyl bromide was added and thesuspension was stirred at r.t. for 2 days. ˜60% of the starting material(45) was converted to the product (46). The reaction was quenched withsat. aq. NaCl and extracted with DCM three times. The combined organiclayer were dried (MgSO₄) and concentrated under vacuum. The residue wastreated with TBAF in THF (1 ml) and THF (1 ml) for 30 min. Evaporationto remove of THF. The residue was dissolved in DCM and aqueous NaHCO₃(sat.) was added. The aqueous layer was extracted with DCM three times.The combined organics was dried over MgSO₄ and concentrated undervacuum. Purification by chromatography on silica (EtOAc to EtOAc:MeOH85:15) gave (46) as a yellow foam (101 mg, 35%). ¹H NMR (d₆ DMSO) δ2.15-2.31 (m, 2H, H-2′), 3.41-3.45 (m, 2H, H-5′), 3.82-3.85 (m, 1H,H-4′), 3.93 (d, J=2.6 Hz, 2H, OCH₂), 4.04-4.06 (m, 1H, H-3′), 4.99 (t,J=5.4 Hz, OH), 5.08-5.24 (m, 2H, ═CH₂), 5.79-5.89 (m, 1H, CH═), 6.15(dd, J=5.9, 9.1 Hz, 1H, H-1′), 6.27 (br s, 2H, NH₂), 7.07 (s, H-8),10.39 (s, 1H, NH). Mass (−ve electrospray) calcd for C₁₄H₁₇IN₄O₄ 432,found 431.

3′-O-Allyl-5′-O-(tert-butyldimethylsilyl)-7-deaza-7-[3-(2,2,2-trifluoroacetamido)-prop-1-ynyl]-2′-deoxyguanosine(47)

Under N₂, a suspension of (46) (104 mg, 0.24 mmol), Pd(PPh₃)₄ (24 mg,0.024 mmol), CuI (9.1 mg, 0.048 mmol), Et₃N (66 μL, 0.48 mmol) andCH≡CCH₂NHCOCF₃ (89 μL, 0.72 mmol) in DMF (2 ml) was stirred at r.t. for15 h. The reaction was protected from light with aluminium foil. AfterTLC indicating the full consumption of starting material, the reactionmixture was concentrated. The residue was diluted with MeOH (20 ml) andtreated with dowex-HCO₃ ⁻. The mixture was stirring for 30 min andfiltered. The solution was concentrated and purified by silica gelchromatography (petroleum ether:EtOAc 50:50 to petroleumether:EtOAc:MeOH 40:40:20) giving (47) as a yellow powder (74 mg, 70%).¹H NMR (d₆ DMSO) δ 2.15-2.39 (m, 2H, H-2′), 3.42-3.44 (m, 2H, H-5′),3.83-3.87 (m, 1H, H-4′), 3.93-3.95 (m, 2H, OCH₂), 4.0-4.07 (m, 1H,H-3′), 4.15 (d, J=5.3 Hz, 2H, ≡CCH₂), 4.91 (t, J=5.4 Hz, OH), 5.08-5.24(m, 2H, ═CH₂), 5.80-5.89 (m, 1H, CH═), 6.15 (dd, J=5.6, 8.9 Hz, 1H,H-1′), 6.28 (br s, 2H, NH₂), 7.24 (s, H-8), 9.98 (t, J=5.3 Hz, 1H, NH),10.44 (s, 1H, NH). Mass (−ve electrospray) calcd for C₁₉H₂₀F₃N₅O₅ 455,found 454.

The nucleoside (47) and proton sponge was dried over P₂O₅ under vacuumovernight. A solution of (47) (73 mg, 0.16 mmol) and proton sponge (69mg, 0.32 mmol) trimethylphosphate (0.5 ml) was stirred with 4 Åmolecular sieves for 1 h. Freshly distilled POCl₃ (18 μl, 0.19 mmol) wasadded and the solution was stirred at 4° C. for 2 h. The mixture wasslowly warmed up to room temperature and bis (tri-n-butyl ammonium)pyrophosphate (1.3 ml, 0.88 mmol) and anhydrous tri-n-butyl amine (0.3ml, 1.28 mmol) was added. After 5 min, the reaction was quenched with0.1 M TEAB (triethylammonium bicarbonate) buffer (10 ml) and stirred for3 h. The water was removed under reduced pressure and the resultingresidue dissolved in concentrated ammonia (ρ 0.88, 10 ml) and stirred atroom temperature for 16 h. The reaction mixture was then evaporated todryness. The residue was dissolved in water and the solution applied toa DEAE-Sephadex A-25 column. MPLC was performed with a linear gradientof 2 L each of 0.05 M and 1 M TEAB. The triphosphate was eluted between0.7 M and 0.8 M buffer. Fractions containing the product were combinedand evaporated to dryness. The residue was dissolved in water andfurther purified by HPLC. t_(r)(48)=20.3 min (Zorbax C18 preparativecolumn, gradient: 5% to 35% B in 30 min, buffer A 0.1 M TEAB, buffer BMeCN). The product (48) was isolated as a white foam (147 O.D., 19.3μmol, 12%, ε₂₆₀=7,600). ¹H NMR (D₂O) δ 2.38-2.46 (m, 2H, H-2′), 3.91 (m,2H, ≡CCH₂), 3.98-4.07 (m, 4H, H-5′, 2H, OCH₂), 4.25 (br s, 1H, H-4′),4.40 (br s, 1H, H-3′), 5.16-5.30 (m, 1H, ═CH₂), 5.83-5.91 (m, 1H, ═CH),6.23-6.27 (m, 1H, H-1′), 7.44 (s, 1H, H-8). ³¹P NMR δ −7.1 (d, J=16.5Hz, 1P, P_(γ)), −10.1 (d, J=19.9 Hz, 1P, P_(α)), −21.5 (t, J=18.0 Hz,1P, P_(β)). Mass (−ve electrospray) calcd for C₁₇H₂₄N₅O₁₃P₃ 599, found598.

7-Deaza-5′-O-diphenylsilyl-7-iodo-2′-deoxyadenosine (49)

TBDPSCl (0.87 g, 2.78 mmol) was added to a stirred solution of7-deaza-7-iodo-2′-deoxyadenosine (1.05 g, 2.78 mmol) in dry pyridine (19ml) at 5° C. under N₂. After 10 min the solution was allowed to rise toroom temperature and stirred for 18 h. The solution was evaporated underreduced pressure and the residue purified by flash chromatography onsilica (DCM to DCM:MeOH 19:1). This gave the desired product (49) (1.6g, 83%). ¹H NMR (d₆ DMSO) δ 1.07 (s, 9H), 2.31-2.36 (m, 1H), 3.76-3.80(dd, 1H, J=11.1, 4.7 Hz), 3.88-3.92 (dd, 1H, J=11.2, 3.9 Hz), 3.97-4.00(m, 1H), 4.49-4.50 (m, 1H), 5.83 (s, 1H), 6.58-6.61 (t, 1H, J=6.7 Hz),7.44-7.55 (m, 6H), 7.68-7.70 (m, 5H), 8.28 (s, 1H). Mass (electrospray)calcd for C₂₇H₃₁IN₄O₃Si 614.12, found 613.

7-Deaza-6-N,N-dimethylformadine-5′-O-diphenylsilyl-7-iodo-2′-deoxyadenosine(50)

A solution of (49) (1.6 g, 2.61 mmol) in MeOH (70 ml) containingdimethylformamide dimethylacetal (6.3 g, 53 mmol) was heated at 45° C.for 18 h. The solution was cooled, evaporated under reduced pressure andpurified by flash chromatography on silica gel (EtOAc to EtOAc:MeOH98:2). This resulted in 1.52 g (87%) of the desired product (50). ¹H NMR(d₆ DMSO) δ 0.85 (s, 9H), 2.05-2.11 (m, 1H), 3.03 (s, 3H), 3.06 (s, 3H),3.53-3.57 (dd, 1H, J=11.1, 4.8 Hz), 3.65-3.69 (dd, 1H, J=11.1, 4 Hz),3.73-3.76 (q, 1H, J=4 Hz), 4.26-4.28 (m, 1H), 5.21-5.22 (d, 1H, J=4.3Hz), 6.39-6.42 (t, 1H, J=6.8 Hz), 7.21-7.32 (m, 6H), 7.46 (s, 1H),7.45-7.48 (m, 4H), 8.15 (s, 1H), 8.68 (s, 1H). Mass (+ve electrospray)calcd for C₃₀H₃₆IN₅O₃Si 669.16, found 670.

3′-O-Allyl-7-deaza-6-N,N-dimethylformadine-5′-O-diphenylsilyl-7-iodo-2′-deoxyadenosine(51)

A solution of (50) (1.52 g, 2.28 mmol) in dry THF (5 ml) was added dropwise at room temperature to a stirred suspension of sodium hydride (60%,109 mg, 2.73 mmol) in dry THF (35 ml). After 45 min the yellow solutionwas cooled to 5° C. and allyl bromide (0.413 g, 3.41 mmol) added. Thesolution was allowed to rise to room temperature and stirred for 18 h.After adding isopropanol (10 drops) the solution was partitioned betweenwater (5 ml) and EtOAc (50 ml). The organic layer was separated and theaqueous solution extracted further with EtOAc (2×50 ml). The combinedorganic solutions were dried (MgSO₄) and evaporated under reducedpressure. The residue was purified by flash chromatography on silica(petroleum ether:EtOAc 1:3 to EtOAc) to give 1.2 g (74%) of the desiredproduct (51) as a gum. ¹H NMR (d₆ DMSO) δ 1.03 (s, 9H), 2.39-2.45 (m,1H), 2.60-2.67 (m, 1H), 3.2 (s, 3H), 3.23 (s, 3H), 3.70-3.74 (dd, 1H,J=11.2, 4.6 Hz), 3.83-3.87 (dd, 1H, J=11, 5.4 Hz), 4.03-4.08 (m, 3H),4.30-4.31 (m, 1H), 5.18-5.21 (m, 1H), 5.28-5.33 (m, 1H), 5.89-5.98 (m,1H), 6.49-6.53 (dd, 1H, J=8.4, 5.8 Hz), 7.41-7.51 (m, 6H), 7.62-7.66 (m,5H), 8.31 (s, 1H), 8.85 (s, 1H). Mass (+ve electrospray) calcd forC₃₃H₄₀IN₅O₃Si 709.19, found 710.

3′-O-Allyl-7-deaza-6-N,N′-dimethylformadine-7-iodo-2′-deoxyadenosine(52)

A 1M solution of TBAF in THF (4.4 ml, 4.4 mmol) was added to a solutionof (51) (1.2 g, 1.69 mmol) in THF (100 ml) at 5° C. under N₂. Thesolution was allowed to rise to room temperature and stirred for 2d. Thesolution was evaporated under reduced pressure and purified by flashchromatography on silica (EtOAc to EtOAc:MeOH 97:3). This gave 593 mg(77%) of the desired product (52). ¹H NMR (d₆DMSO) δ 2.54 (m, 2H), 3.40(s, 3H), 3.44 (s, 3H), 3.72-3.8 (m, 2H), 4.18-4.21 (m, 1H), 4.23-4.27(m, 3H), 4.4-4.42 (d, 1H, J=5.7 Hz), 5.35-5.41 (m, 2H), 5.49-5.5 (q, 1H,J=1.7 Hz), 5.53-5.55 (q, 1H, J=1.7 Hz), 6.1-6.2 (m, 1H), 6.67-6.70 (dd,1H, J=8.8, 5.5 Hz), 7.96 (s, 1H), 8.53 (s, 1H), 9.06 (s, 1H). Mass (−veelectrospray) calcd for C₁₇H₂₂IN₅O₃ 471.08, found 472.

3′-O-Allyl-7-deaza-7-iodo-2′-deoxyadenosine (53)

A solution of (52) (593 mg, 1.3 mmol) in MeOH (20 ml) containing 35%aqueous ammonia (20 ml) was heated at 50° C. for 2d. After cooling thesolution was evaporated under reduced pressure and then azeotroped withtoluene (3×10 ml). This resulted in 530 mg (98%) of the desired product(53) as a solid. ¹H NMR (d₆ DMSO) δ 2.39 (m, 1H), 3.56-3.65 (m, 2H),4.03-4.05 (m, 1H), 4.09-4.11 (m, 2H), 5.23-5.25 (d, 1H, J=10.6 Hz),5.35-5.4 (d, 1H, J=15.4 Hz), 5.95-6.05 (m, 1H), 6.48-6.51 (dd, 1H,J=8.9, 5.5 Hz), 6.6-6.95 (s, 1H), 7.75 (s, 1H), 8.16 (s, 1H). Mass (+veelectrospray) calcd for C₁₄H₁₇IN₄O₃ 416.03, found 417.

3′-O-Allyl-7-deaza-7-[3-(2,2,2-trifluoroacetamide)]-2′-deoxyadenosine(54)

To a solution of (53) (494 mg, 1.19 mmol) in dry DMF (17 ml) was addedsequentially copper (I) iodide (45.1 mg, 0.24 mmol),N-2,2,2-trifluoro-N-prop-2-ynylacetamide (538 mg, 3.56 mmol), Et₃N (240mg, 2.38 mmol) and Pd(Ph₃P)₄ (137 mg, 0.12 mmol) at room temperature.The flask was wrapped in foil to exclude light and stirred under N₂ for18 h. Then MeOH (10 ml) and a small spatula of dowex bicarbonate H⁺ formwere added and the mixture stirred for 30 min. The mixture was filtered,evaporated under reduced pressure and the residue triturated with MeOHto remove palladium salts. The filtrate was evaporated under reducedpressure and purified by flash chromatography on silica (DCM to DCM:MeOH97:3). The desired product (54) was obtained as brown solid (490 mg,94%). ¹H NMR (d₆ DMSO) δ 2.25-2.31 (m, 1H), 2.98-3.04 (m, 1H), 3.41-3.49(m, 2H), 3.88-3.95 (m, 3H), 4.10-4.12 (d, 1H, J=5.2 Hz), 4.22-4.23 (d,2H, J=5.3 Hz), 5.07-5.12 (m, 2H), 5.19-5.24 (dd, 1H, J=17.3, 1.9 Hz),5.79-5.89 (m, 1H), 6.31-6.35 (dd, 1H, J=8.6, 5.6 Hz), 7.69 (s, 1H), 8.02(S, 1H). Mass (−ve electrospray) calcd for C₁₉H₂₀F₃N₅O₄ 439.15, found438.

3′-O-Allyl-7-[3-aminoprop-1-ynyl]-7-deaza-2′-deoxyadenosine5′-O-nucleoside triphosphate (55)

The nucleoside (54) and proton sponge was dried over P₂O₅ under vacuumovernight. A solution of (54) (84 mg, 0.191 mmol) and proton sponge (49mg, 0.382 mmol) in trimethylphosphate (600 μl) was stirred with 4 Åmolecular sieves for 1 h. Freshly distilled POCl₂ (36 μl, 0.388 mmol)was added and the solution was stirred at 4° C. for 2 h. The mixture wasslowly warmed up to room temperature and bis (tri-n-butyl ammonium)pyrophosphate 0.5 M in solution in DMF (1.52 ml, 0.764 mmol) andanhydrous tri-n-butyl amine (364 μl, 1.52 mmol) was added. After 5 min,the reaction was quenched with 0.1 M TEAB (triethylammonium bicarbonate)buffer (5 ml) and stirred for 3 h. The water was removed under reducedpressure and the resulting residue dissolved in concentrated ammonia (p0.88, 5 ml) and stirred at room temperature for 16 h. The reactionmixture was then evaporated to dryness. The residue was dissolved inwater and the solution applied to a DEAE-Sephadex A-25 column. MPLC wasperformed with a linear gradient of 0.05 M to 1 M TEAB. Fractionscontaining the product were combined and evaporated to dryness. Theresidue was dissolved in water and further purified by HPLC. HPLC:t_(r)(55)=: 22.60 min (Zorbax C18 preparative column, gradient: 5% to35% B in 20 min, buffer A 0.1M TEAB, buffer B MeCN) The product wasisolated as a white foam (17.5 μmol, 5.9%, ε₂₈₀=15000). ¹H NMR (D₂O) δ2.67-2.84 (2m, 2H, H-2′), 4.14 (br s, 2H, CH₂NH), 4.17-4.36 (m, 2H,H-5′), 4.52 (br s, 1H, H-4′), 6.73 (t, J=6.6 Hz, 1H, H-1′), 8.06 (s, 1H,H-8), 8.19 (s, 1H, H-2). ³¹P NMR (D₂O) δ −5.07 (d, J=21.8 Hz, 1P,P_(γ)), −10.19 (d, J=19.8 Hz, 1P, P_(α)), −21.32 (t, J=19.8 Hz, 1P,P_(β)). Mass (−ve electrospray) calcd for C₁₅H₂₁N₈O₁₂P₃ 598.05, found596

To the Cy3 disulphide linker (2.6 μmol) in solution in DMF (450 μl) isadded at 0° C. 100 μl of a mixture of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,1-hydroxybenzotriazole hydrate and N-methylmorpholine (26 μM each) inDMF. The reaction mixture was stirred at room temperature for 1 h. Thereaction was monitored by TLC (MeOH:CH₂Cl₂ 4:6) until all the dye linkerwas consumed. Then 400 μl of DMF are added at 0° C., followed by thenucleotide (55) (3.9 μmol), in solution in water (100 μl) and thereaction mixture and stirred at room temperature overnight. TLC(MeOH:CH₂Cl₂ 4:6) showed complete consumption of the activated ester anda dark red spot appeared on the baseline. The reaction was quenched withTEAB buffer (0.1M, 10 ml) and loaded on a DEAE Sephadex column (2×5 cm).The column was first eluted with 0.1 M TEAB buffer (100 ml) to wash offorganic residues and then 1 M TEAB buffer (100 ml). The desiredtriphosphate (56) was eluted out with 1 M TEAB buffer. The fractioncontaining the product were combined, evaporated and purified by HPLC.HPLC conditions: t_(r)(56)=: 21.38 min (Zorbax C18 preparative column,gradient: 5% to 15% B in 1 min, then 4 min at 15% B, then 15 to 35% B in15 min, buffer A 0.1M TEAB, buffer B MeCN). The product was isolated asdark pink solid (0.15 μmol, 12.5%, ε₅₅₀=15000). ¹H NMR (D₂O) δ 2.03 (t,J=6.4 Hz, 2H, CH₂), 2.21-2.33 (m, 1H, H-2′), 2.37-2.49 (m, 1H, H-2′),2.50 (t, J=6.3 Hz, 2H, CH₂), 2.66 (t, J=5.4 Hz, 2H, CH₂), 3.79 (t, J=6.4Hz, 2H CH₂), 3.99 (m, 4H, CH₂N, H-5′), 4.18 (br s, 1H, H-4′), 6.02, 6.17(2d, J=13.6 Hz, 2H, H_(ar)) 6.30 (dd, J=6.1, 8.6 Hz, H-1′), 7.08, 7.22(2d, J=7.8, 8.6 Hz, 2H, 2×═CH), 7.58-7.82 (m, 6H, 2H_(Ar), H-2, H-8),8.29 (t, J=13.6 Hz, ═CH). ³¹P NMR (D₂O) δ −4.83 (m, 1P, P_(γ)), −10.06(m, 1P, P_(α)), −20.72 (m, 1P, P_(β)).

Cleavage of 3′-Allyl Group in Aqueous Conditions

The following shows a typical deblocking procedure for a 3′blockednucleoside in which approximately 0.5 equivalents of Na₂PdCl₄ and 4equivalents of the water-soluble phosphine ligand L were employed, inwater, at 50° C. Tfa stands for trifluoracetyl:

To a solution of Ligand L (7.8 mg, 13.7 μmol) in degassed H₂O (225 μl)was added a solution of Na₂PdCl₄ (0.5 mg, 1.6 μmol) in degassed H₂O (25μl) in an eppendorff vial. The two solutions were mixed well and after 5min a solution of B (1 mg, 2.3 μmol) in H₂O (250 μl) was added. Thereaction mixture was then placed in a heating block at 50° C. Thereaction could be followed by HPLC. Aliquots of 50 μl were taken fromthe reaction mixture and filtered through an eppendorff filter vial(porosity 0.2 μm); 22 μl of the solution were injected in the HPLC tomonitor the reaction. The reaction was purified by HPLC. In a typicalexperiment the cleavage was finished (i.e. >98% cleavage had occurredafter 30 min).

3′-OH Protected with a 3,4 Dimethoxybenzyloxymethyl Group as a ProtectedForm of a Hemiacetal

Nucleotides bearing this blocking group have similar properties to theallyl example, though incorporate less rapidly. Deblocking can beachieved efficiently by the use of aqueous buffered cerium ammoniumnitrate or DDQ, both conditions initially liberating the hemiacetal (1)which decomposes to the required (2) prior to further extension:

The 3′-OH may also be protected with benzyl groups where the phenylgroup is unsubstituted, e.g. with benzyloxymethyl, as well as benzylgroups where the phenyl group bears electron-donating substituents; anexample of such an electron-rich benzylic protecting group is3,4-dimethoxybenzyloxymethyl.

In contrast, electron-poor benzylic protecting groups, such as those inwhich the phenyl ring is substituted with one or more nitro groups, areless preferred since the conditions required to form the intermediategroups of formulae —C(R′)₂—OH, —C(R′)₂—NH₂, and —C(R′)₂—SH aresufficiently harsh that the integrity of the polynucleotide can beaffected by the conditions needed to deprotect such electron-poorbenzylic protecting groups.

3′-OH Protected with a Fluoromethyloxymethyl Group as a Protected Formof a Hemiacetal

—O—CH₂—F

Nucleotides bearing this blocking group may be converted to theintermediate hemiacetal using catalytic reactions known to those skilledin the art such as, for example, those using heavy metal ions such assilver.

1. (canceled)
 2. (canceled)
 3. A composition comprising: a planar solidsupport; a plurality of different target polynucleotides immobilized atdistinct regions on the solid support, wherein the distinct regionscomprise multiple copies of one of the plurality of different targetpolynucleotides; a plurality of different complementary oligonucleotideshybridized to the different target polynucleotides; and an aqueoussolution contacting the solid support, wherein the aqueous solutioncomprises: at least 75% by volume water as a continuous phase of theaqueous solution; at least one fluorescent label; and a water-solubletri-C₁₋₆-alkyl phosphine substituted with a plurality of hydroxyl,carboxyl, carboxylate, amino, or sulfonate groups.
 4. The composition ofclaim 3, wherein the aqueous solution comprises a plurality of differentfluorescent labels.
 5. The composition of claim 4, wherein thefluorescent labels comprise fluorescein, rhodamine, alexa, bodipy,acridine, coumarin, pyrene, benzanthracene cyanine, or a mixturethereof.
 6. The composition of claim 5, wherein the fluorescent labelscomprise Cy3 or Cy5.
 7. The composition of claim 3, wherein the aqueoussolution comprises about 95% by volume of water.
 8. The composition ofclaim 7, wherein the aqueous solution comprises greater than 98% byvolume of water.
 9. The composition of claim 3, wherein thewater-soluble tri-C₁₋₆-alkyl phosphine has a plurality of hydroxylgroups.
 10. The composition of claim 3, wherein the water-solubletri-C₁₋₆-alkyl phosphine has the structure of:

or a salt thereof.
 11. The composition of claim 10, wherein the salt ofthe water-soluble tri-C₁₋₆-alkyl phosphine is a trisodium salt.
 12. Thecomposition of claim 3, wherein the planar solid support comprises aglass surface, a silicon surface, a ceramic surface, or a plasticsurface.
 13. The composition of claim 12, wherein the planar solidsupport comprises fabricated arrays of oligonucleotides.
 14. Thecomposition of claim 3, wherein the aqueous mixture has a temperature ofabout 50° C. or higher.
 15. The composition of claim 14, wherein theaqueous mixture has a temperature of about 50° C. to about 80° C. 16.The composition of claim 3, wherein the label in aqueous mixturecomprises a multi-component label.
 17. The composition of claim 14,wherein the multi-component label comprises a fluorescent antibody. 18.An oligonucleotide comprising a nucleotide residue, wherein saidnucleotide residue comprises the structure:

wherein Z is —CH₂N₃; B is a nucleobase selected from the groupconsisting of a purine, a pyrimidine and a deazapurine; and

comprises


19. The oligonucleotide of claim 18, wherein the nucleobase is selectedfrom the group consisting of

wherein the asterisk * indicates the attachment point to the sugarmoiety of the nucleotide residue.
 20. The oligonucleotide of claim 18,wherein the oligonucleotide is hybridized to a target polynucleotide.21. The oligonucleotide of claim 20, wherein the target polynucleotideis immobilized to a solid support.
 22. The oligonucleotide of claim 21,wherein the solid support comprises an array of immobilized targetpolynucleotides.